Method for reducing carryover contamination in an amplification procedure

ABSTRACT

The present invention provides an efficient and economical method for reducing carryover contamination in an amplification procedure. The method of the present invention enables background caused by contaminant amplification product to be reduced or eliminated through the incorporation of at least one modification into the amplification product. The modified amplification product is readily distinguishable from the target sequence in a test sample. Prior to amplifying the target in a new test sample, the sample may be treated to selectively eliminate the contaminant amplification product so that it cannot be amplified in the new sample.

This application is a continuation of application Ser. No. 08/057,192,filed May 03, 1993, now U.S. Pat. No. 5,427,929, which is herebyincorporated by reference, which is a continuation of application Ser.No. 07/686,478 filed Apr. 9, 1991, now abandoned, which is acontinuation-in-part of application Ser. No. 07/517,631 filed May 1,1990, now abandoned.

BACKGROUND

Diagnostic assays are routinely used to detect the presence and/orquantity of analytes in test samples taken from a patient or othersubject. Typical analytes include antigens and antibodies, which aremeasured using immunodiagnostic techniques to identify certain diseasestates and various types of non-disease conditions, such as pregnancy.The achievement of high levels of sensitivity and specificity isimportant in most diagnostic assays. This is particularly true where theanalytes of interest are present at relatively low concentrations.Various improvements which have enabled the attainment of higher levelsof sensitivity in immunodiagnostic procedures have included the use ofmonoclonal antibodies in the assay configurations and the incorporationof methods for amplifying the signal used as a tag in these types ofassays.

More recently, advances in the field of molecular biology have enabledthe detection of specific nucleic acid sequences in test samples using atechnique known as probe diagnostics. In probe diagnostics, a nucleicacid sequence is used to "probe" the sample by specifically binding toits complementary nucleic acid target analyte. This makes it possible todetect diseases at an early stage, because the nucleic acid geneticmaterial often appears in a test sample months, or even years, beforesufficient time has elapsed for the nucleic acid target to betranscribed into an antigen. This is particularly true in certain typesof sexually transmitted diseases, such as infection by the humanimmunodeficiency virus. Ranki et al, The Lancet, 85(59) 589-593 (1987).In addition to the detection of various diseases and genetic disorders,the ability of probe diagnostics to identify the presence of specificgenes can also be used to obtain other pertinent genetic information,such as the presence of genes coding for antigens responsible for graftrejection, as well as genetic information used in cancer and oncogenetesting and in forensic medicine.

At its full potential, probe diagnostics are theoretically capable ofdetecting as little as one molecule in a test sample. One of the majorobstacles to achieving the full potential of probe diagnostics is theinherent difficulty which is encountered in detecting the extremelyminute quantities of target sequences often present in test samples. Asa consequence, recent efforts to improve the sensitivity of probediagnostics have centered around methods for amplifying the targetnucleic acid sequence. Amplification of the target sequence may beaccomplished in any one of a number of ways involving the repetitivereproduction or replication of the given DNA or RNA target nucleic acidsequence, resulting in linear or exponential amplification, dependingupon the particular method used.

Early methods which were used routinely for production of multiplecopies of nucleic acid sequences involved cloning the target nucleicacid sequence into an appropriate host cell system. These methods employtraditional cloning techniques wherein the desired nucleic acid isinserted into an appropriate vector which is subsequently used totransform the host. When the host is grown in culture, the vector isreplicated, producing additional copies of the desired nucleic acidsequence. The target nucleic acid sequence which is inserted into thevector can be either naturally occurring or synthesized. In other words,the desired target nucleic acid sequence can be synthesized in vitro andthen inserted into a vector which is amplified by growth, as disclosedin U.S. Pat. No. 4,293,652.

U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose an automat able method,commonly referred to as polymerase chain reaction (PCR), for amplifyingthe amount of target nucleic acid sequence in a test sample. PCRamplification utilizes two oligonucleotide primers which arecomplementary to the ends of different portions on opposite strands of asection of the target sequence. Following hybridization of these primersto the target, extension products complementary to the target sequenceare formed in the presence of DNA polymerase and an excess of nucleosidetriphosphates. The primers are oriented so that DNA synthesis by thepolymerase proceeds across and through the region between the primers.The hybridized extension product is then denatured from the target andthe cycle repeated, with extension product also acting as template forthe formation of additional extension product in subsequent cycles ofamplification. Cycling continues until a sufficient quantity of thetarget nucleic acid sequence is produced to result in measurable signalin the assay of choice. Each successive cycle theoretically doubles theamount of nucleic acid synthesized in the previous cycle, resulting inexponential accumulation of amplified product.

International Publication No. WO 89/12696 discloses a different type ofautomat able amplification procedure. In this type of amplificationprocedure, presynthesized pairs of amplification probes hybridizecontiguously to a section of the target sequence. The contiguous endsare then ligated to form the complementary amplification product.Following ligation, the completed amplification product is separatedfrom the target by heat denaturation. The process is then repeated, withboth the target and resulting amplification product acting as a templatefor the probes in subsequent cycles, until a sufficient quantity of thetarget nucleic acid sequence is produced to result in measurable signalin the selected assay. As with PCR, each successive cycle theoreticallydoubles the amount of nucleic acid from a previous cycle. Amplificationmethods employing presynthesized probes have generally been referred toas ligase chain reaction (LCR), although ligation of the probes can beachieved by means other than the action of a ligase, such as, forexample, a chemical or photochemical ligation.

Although nucleic acid amplification methods have revolutionized probediagnostics by enabling the detection of extremely small quantities ofnucleic acid sequences in test samples, they have also created their ownproblem in the routine diagnostic setting, namely, one of falsepositives due to carryover contamination. The repeated amplification ofa nucleic acid analyte to many millions or billions of times its normalconcentration in a test sample raises the possibility of carryovercontamination of new samples from the samples containing amplifiedtarget. This, in turn, can create artificially high signals insubsequent test samples, including false positives where negativesamples are contaminated.

Carryover contamination may occur as the result of mechanical carryoverfrom sample to sample or as the result of airborne contamination.Airborne contamination is unavoidable where reaction vessels containingamplified test sample are opened for any reason, such as for theaddition of reagents or for sampling of the amplified analyte fordetection. This act alone can aspirate millions of molecules ofamplification product into the air, contaminating a normal laboratorywork area with hundreds of molecules per cubic inch. Thus far, it hasbeen impossible to completely guard against the contamination of otherspecimens through contact with these airborne copies, regardless of thedegree of care exercised by the operator(s).

The following techniques for dealing with the contamination problemscreated by amplification of target sequences, although suggested for usein a PCR type of amplification procedure, would be generally applicableto other types of amplification procedures: (1) physical separation(such as separate rooms) of pre-amplification and post-amplificationsamples; (2) separate storage and aliquotting of reagents; (3) the useof positive displacement pipettes; (4) meticulous laboratory technique;and, (5) cautious selection of controls. Amplifications--A forum for PCRUsers, 2, 4 (1989). These measures, however, are not only costly, butassume the most ideal of conditions. Furthermore, even if theseexpensive techniques are practiced fastidiously, precautions such asthese cannot completely eliminate the contamination problem. Thereremain inherent difficulties from laboratory workers who invariablycarry contamination on their bodies and clothes and from the circulationof contaminated air from room to room through air vents. Kitchin,Nature, 344, 201 (1990).

Treatment of reagents with ultraviolet light has been suggested for thecontrol of contaminant amplification product in a PCR type ofamplification procedure. This suggestion is based on the known abilityof ultraviolet light to destroy the integrity of DNA. Although themechanism for this action is unknown, it has been demonstrated thatPCR-based contaminant amplification product can be destroyed in buffersas well as in primer, dNTP, and Taq polymerase preparations byirradiation with ultraviolet light. Sarkar et al, Nature, 343, 27(1990). Although the single-stranded PCR primers are apparently able tosurvive this treatment, double-stranded pairs of LCR probes are likelyto be more sensitive to the irradiation treatment, and may be destroyed.Furthermore, irradiation with ultraviolet light cannot be used directlyon unassayed test samples, because this would result in the destructionof double-stranded target molecules by the irradiation treatment.

It is an objective of the present invention to provide a cost effectivemethod for significantly reducing the carryover contaminationencountered when using amplification procedures in diagnostic probeassays. It is a further objective of the present invention to provide acontamination reduction method which is simple to perform and which isadaptable to a number of different types of amplification procedures.

SUMMARY OF THE INVENTION

The present invention provides an efficient and economical method forreducing amplification product contamination in an amplificationprocedure. The method of the present invention is carried out bymodifying the amplification product such that the modified amplificationproduct is distinguishable from the target sequence. Prior to amplifyingthe target nucleic acid in a new test sample, the new sample may betreated in an appropriate manner to selectively remove, destroy, orotherwise render the modified contaminant amplification productnonviable so that it cannot be amplified in subsequent amplificationprocedures. Treatment in this way reduces carryover contamination, andthe false positives caused by this type of contamination, withoutaffecting levels of the naturally occurring target sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing ligand modification of an LCR-derivedamplification product and subsequent removal of the modifiedamplification product with immobilized specific binding partner.

FIG. 2 is a diagram showing ligand modification of a PCR-derivedamplification product and subsequent removal of the modifiedamplification product with immobilized specific binding partner.

FIG. 3 is a diagram showing cross-linking agent modification of anLCR-derived amplification product and subsequent irreversiblecross-linking of the modified amplification product.

FIG. 4 is a diagram showing cross-linking agent modification of aPCR-derived amplification product and subsequent irreversiblecross-linking of the modified amplification product.

FIG. 5 is a diagram showing restriction site modification of anLCR-derived amplification product and subsequent cleavage of themodified amplification product with a restriction enzyme.

FIG. 6 is a diagram showing restriction site modification of aPCR-derived amplification product and subsequent cleavage of themodified amplification product with a restriction enzyme.

FIG. 7 is a diagram showing partial priming of a portion of restrictionsite modified PCR-derived amplification product which has been cleavedwith a restriction enzyme.

FIG. 8 is a diagram showing restriction site modification of aPCR-derived amplification product and subsequent cleavage of themodified amplification product with a remote cutting restriction enzyme.

FIG. 9 is a diagram showing restriction site modification of anLCR-derived amplification product and subsequent cleavage of themodified amplification product with a remote cutting restriction enzyme.

FIG. 10 shows the preparation of oligonucleotide amplification probes orprimers incorporating a chemically cleavable site.

FIG. 11 shows the amplification sequence, amplification probes,resulting amplification product, and detection probes from Examples 1and 2.

FIG. 12 is a photograph of an autoradiogram showing the relativeamplification efficiencies of target and restriction enzyme modifiedamplification product following treatment of both the target sequenceand the modified amplification product with restriction enzyme.

FIG. 13 shows the polysite DNA used in Example 3 to evaluate remotecutting restriction enzymes.

FIG. 14 is a photograph of an autoradiogram showing the relative cuttingefficiencies of various remote cutting restriction enzymes with respectto the polysite DNA.

FIG. 15 is a diagram showing double restriction site modification of aPCR-derived amplification product and subsequent cleavage of themodified amplification product with a remote cutting restriction enzyme.

FIG. 16 shows a 147 base pair amplification sequence (contained in pUC9), corresponding modified and unmodified PCR amplification primers, anda detection primer from Example 4.

FIG. 17 is a photograph of an autoradiogram showing the relativeefficiencies of PCR amplification using modified and unmodified primers,as well as the inability of the treated modified amplification productto serve as template in subsequent amplifications, as demonstrated inExample 4.B. and 4.C.

FIG. 18 is a photograph of an autoradiogram showing the effectivedestruction of a modified PCR-derived amplification product by treatmentwith the corresponding cutting agent, as demonstrated in Example 4.C.

FIG. 19 is a diagram showing the products resulting from single anddouble cuts on a PCR amplification product containing two remote cuttingrestriction site modifications.

FIG. 20 shows a 162 base pair HIV amplification sequence, correspondingmodified amplification primers and a detection primer from Example 5.

FIG. 21 is a photograph of an autoradiogram showing the formation andquantitation of PCR-derived modified amplification product, asdemonstrated in Example 5.A.

FIG. 22 is a photograph of an autoradiogram showing the effective"pre-amplification" destruction of carryover contamination in a PCR typeof amplification procedure, as demonstrated in Example 5.B.

FIG. 23 is a diagram showing a 75 base pair HIV amplification sequence,two ribonucleotide modified PCR amplification primers, and a detectionprimer used in Example 6.

FIG. 24 is a photograph of an autoradiogram showing PCR amplificationusing PCR primers containing a single ribonucleotide substitution ontheir respective 3'-ends, as demonstrated in Example 6.A.

FIG. 25 is a photograph of an autoradiogram showing he quantitation ofribonucleotide modified PCR-derived amplification product by comparisonto standards, as demonstrated in Example 6.A.

FIG. 26 is a photograph of an autoradiogram showing the quantitativedestruction of ribonucleotide modified PCR-derived amplification productusing RNAse A as a cutting agent, as demonstrated in Example 6.B.

FIG. 27 is a photograph of an autoradiogram showing the effective"pre-amplification" destruction of ribonucleotide modified PCR-derivedamplification product using strong base as a cutting agent, asdemonstrated in Example 6.C.

FIG. 28 is a diagram showing a 45 base pair HIV amplification sequenceand the corresponding ribonucleotide modified amplification probes usedto generate ribonucleotide modified LCR-derived amplification product inExample 7.

FIG. 29 is a photograph of an autoradiogram showing LCR amplificationusing amplification probes containing a single ribonucleotidesubstitution on their respective 3'-ends, as demonstrated in Example7.A.

FIG. 30 is a photograph of an autoradiogram showing the quantitation ofribonucleotide modified LCR-derived amplification product by comparisonto standards, as demonstrated in Example 7.A.

FIG. 31 is a photograph of an autoradiogram showing the effective"pre-amplification" destruction of ribonucleotide modified LCR-derivedamplification product using strong base as a cutting agent, asdemonstrated in Example 7.B.

FIG. 32 is a photograph of an autoradiogram showing the quantitativedestruction of ribonucleotide modified LCR-derived amplification productusing RNAse A as a cutting agent, as demonstrated in Example 7.C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention enables background caused by contaminantamplification product to be reduced or eliminated through theincorporation of at least one modification into the amplificationproduct. The modified amplification product is readily distinguishablefrom the target sequence in a test sample, and may be selectivelyeliminated.

In order to more clearly understand the invention, it will be useful toset forth the definitions of certain terms that will be used herein:

Amplification means increasing the number of copies of a nucleic acidsequence in a test sample. The copies which are generated duringAmplification may be exact copies or complementary copies. In addition,the copies may be modified by a means for controlling contamination.Amplification may proceed in a linear manner or in an exponentialmanner; i.e., at a rate greater than linear Amplification.

Nucleic Acid Sequence is a deoxyribonucleotide or a ribonucleotide whichmay be modified with respect to: (1) the phosphate backbone; (2) thenucleosides; and/or, (3) the sugar moiety of the oligonucleotide.Nucleic Acid Sequences can contain labels or other attached moieties andcan be interrupted by the presence of still other moieties, as long ashybridization can occur.

Target Sequence is the nucleotide sequence being sought in a particularassay.

Amplification Sequence is a designated length of the target sequencewhich initially acts as template sequence in an amplification procedure.The Amplification Sequence may comprise the entire length of the targetsequence or a representative portion thereof.

Template Sequence is the nucleic acid sequence upon which amplificationproduct is formed. In the first cycle of amplification, theamplification sequence acts as the Template Sequence. In subsequentcycles of amplification, amplification product also serves as a TemplateSequence.

Amplification Probe is a nucleic acid sequence which is either: (1)complementary to a portion of a single strand of a double-strandedamplification sequence; or, (2) complementary or identical to a portionof a single-stranded amplification sequence. The Amplification Probeshybridize to the amplification sequence sufficiently adjacent to eachother to enable the probes to be joined together. The AmplificationProbe may or may not be modified at one or both ends for joining toother Amplification Probes. In addition, an Amplification Probe may ormay not be modified by the incorporation of a means for controllingcontamination.

Amplification Primer as used herein refers to a nucleic acid sequencewhich is complementary to an end portion of an amplification sequenceand which is capable of acting as a point of initiation of synthesis ofa primer extension product which is complementary to the amplificationsequence. The primer extension product is formed in the presence ofnucleotides and an agent for polymerization such as DNA polymerase. AnAmplification Primer may or may not be modified by the incorporation ofa means for controlling contamination.

Presynthesized probe or primer as used herein means an oligonucleotidesequence which has been synthesized prior to being added to a testsample reaction mixture.

Extending End of an amplification primer means the end of theamplification primer which is acted on by a polymerase to form extensionproduct. The Extending End will be the 3'-end.

Amplification Product refers to the like copy and/or the complementarycopy of an amplification sequence. The Amplification Product issynthesized in situ during an amplification procedure. AmplificationProduct may be the ligated nucleic acid sequence which is produced fromligation of a series of amplification probes which are contiguouslyhybridized to an amplification sequence. Amplification Product may alsobe the extension product of a polymerase chain reaction. The termAmplification Product includes modified amplification product.

Modified Amplification Product as used herein refers to amplificationproduct which contains at least one modification site.

Modification Site refers to a single location into which has beenincorporated a means for controlling amplification product contaminationon an amplification product. A Modification Site includes, for examplebut without limitation, the introduction of a ligand, the introductionof an cross-linking agent or chemically cleavable site, and basechange(s) to achieve an enzyme recognition site.

Contaminant Amplification Product is amplification product which isintroduced into a test sample by a means other than amplification of theamplification sequence originally present in a test sample. TheContaminant Amplification Product may, for example, be the result ofmechanical carryover or the result of airborne carryover contaminationof a test sample with amplification product from a previously amplifiedsample or samples.

Complementary refers to sufficient complementarity to enablehybridization to occur. Complete complementarity is not required.

Substantially Complementary refers to complementarity wherein at leastone base is mismatched.

Pseudo Restriction Site is a sequence of nucleic acid residues which butfor the alteration of at least one nucleotide would represent arestriction enzyme recognition site. A Pseudo Restriction Site is notcleaved by a restriction enzyme that would recognize the unalteredsequence. Although the term "alteration" is used, Pseudo RestrictionSites are naturally occurring, and it will be appreciated that anynucleotide sequence not representing a restriction site will be a PseudoRestriction Site.

A Preferred Pseudo Restriction Site is a pseudo restriction site whichrequires only one base modification to achieve a restriction enzymerecognition site.

Recognition Site as used herein means the specific sequence recognizedby an enzyme, such as a restriction endonuclease or RNAse.

Enzyme Cleavage Site means the phosphodiester bond which is hydrolyzedby an enzyme, such as a restriction endonuclease or RNAse.

Remote Cutting Restriction Endonuclease, or Remote Cutter, is arestriction endonuclease that cleaves double-stranded DNA at a siteoutside of the enzyme recognition site.

The present invention is directed to a means for controlling carryovercontamination from contaminant amplification product. A modifiedamplification product is created, as part of the amplificationprocedure, by incorporating at least one modification site into theamplification product such that the modified amplification product isdistinguishable from target sequence. As a result of this modification,the modified contaminant amplification product can be selectivelyeliminated by removing, destroying, or otherwise rendering the modifiedamplification product nonviable as template sequence for subsequentamplification events. The means for selective elimination of modifiedamplification product will, of course, vary with respect to theparticular modification incorporated into the amplification product.

It is possible to incorporate the contamination control method of thepresent invention into a pre-amplification treatment, apost-amplification treatment, or a combination of both pre- andpost-amplification treatment of test samples. In the case ofpost-amplification treatment, modified amplification product isselectively eliminated from amplified test samples after the completionof the amplification procedure. This effectively minimizes the spread ofthe modified product throughout the laboratory or work space, where itcan contaminate new test samples, thus producing false positives.Post-amplification treatment does not, however, completely eliminate thecontamination problem, because some degree of airborne contaminationwill still occur through the simple process of opening the reaction tubeto add the reagents (e.g., cutting agent) necessary to selectivelyeliminate the contaminant product.

In most cases, however, it will be possible, and in fact preferred, to"pre-treat" new test samples to selectively eliminate contaminantmodified amplification product prior to subjecting the new test samplesto amplification. In the case of pre-amplification treatment, modifiedamplification product may contaminate new test samples, with thecontaminant amplification product being substantially completely removedor destroyed within the new test sample itself. Where the amplificationreagents (e.g., amplification probes or primers) are susceptible todestruction by the same agent used to selectively eliminate thecontaminant amplification product, it will be necessary to add theamplification reagents following pre-amplification treatment of the testsample. Where, however, the amplification reagents are resistant todestruction by the agent used to selectively eliminate the contaminantproduct (e.g., where certain types of remote restriction enzymerecognition modification sites or ribonucleotide substitutions areused), the destructive agent can be added to the test sample immediatelyprior to amplification; i.e., after all of the necessary reagents havebeen added to the test sample, thus providing the greatest degree ofcontamination control.

The method of the present invention enables carryover contamination tobe controlled in an efficient, reliable, and economical manner. This isparticularly important in a diagnostic setting where amplificationprocedures are routinely conducted. In these types of settings the sameanalyte is continually amplified over and over again, furtherexacerbating the problem of amplification product contamination of newsamples. The longer the period of time over which a particular analyteis assayed, the greater the contamination problem. For example, theaspiration of only one μl from a single 100 μl sample, which has beenamplified to 100 femtomoles of amplification product, will release 600million copies of target into the air. If evenly dispersed in a typicalwork environment, this results in a concentration of approximately 350molecules of contaminant amplification product for each cubic inch ofwork area.

The solution to carryover contamination provided by the presentinvention does not require the often elaborate and painstaking steps ofprior art methods. For example, the present invention eliminates theneed for the use of separate rooms for the introduction, amplification,and detection of new test samples. Likewise, the use of disposableclothes, costly positive displacement pipettes, and special disposableplumbing and sample handling devices are also unnecessary. The methodprovided herein has less inherent variation and does not contain theself-imposed limitations of prior art methods such as, for example,limitation of the amount of standard that is run alongside anamplification procedure. Many other advantages of the method of thepresent invention will be apparent to those skilled in the art.

The method of the present invention contemplates and embraces theintroduction of a number of different types of modifications into theamplification product which is generated in an amplification procedure.What is important in the selection of a particular modification is thatthe modification provide a means for distinguishing and/or separating ordestroying the amplification product from the target sequence insubsequently run test samples. In this way, contaminant amplificationproduct is removed, destroyed, or otherwise rendered nonviable as atemplate for amplification prior to amplification of the target sequencein the treated new test sample.

The types of modifications useful in distinguishing contaminantamplification product will be apparent to those skilled in the art basedupon the teachings of the present invention. These modifications mayinclude, for example, the introduction of a ligand, the introduction ofa cross-linking agent, or the introduction of an enzyme recognition site(including restriction enzyme recognition sites) or other suitablecleavable moiety. Certain of the modifications of the present inventionwill have a greater number of limitations than others, as will be morefully described. As a consequence, certain modifications will bepreferred in certain situations, depending upon the characteristics of aparticular analyte. The preferred modification for an amplificationproduct in a given assay procedure will be apparent to persons ofordinary skill in the art, based upon the characteristics of the analyteto be amplified and the teachings of the present invention.

A modification is preferably incorporated into an amplification productby using presynthesized amplification probes or primers which containthe selected modification. Amplification with these modified probes orprimers will, in turn, incorporate the modification(s) into thecompleted amplification product(s). Modification of the amplificationprobes or primers may be accomplished by any one of a number of methodsthat are known to those skilled in the art. In the case of ligands andother similar types of modifications, for example, the modification canbe introduced into the completed oligonucleotide probe or primerfollowing synthesis of the oligonucleotide. In the case of enzymerecognition sites, the modification(s) can simply be substituted intothe oligonucleotide probe or primer during synthesis. It may also bepossible, in the case of PCR, to amplify the target with polymerase inthe presence of one or more modified nucleoside triphosphates togenerate modified amplification product. (See, for example, Langer etal, Proc. Natl. Acad. Sci. USA, 78(11), 6633-6637 (1981).

The number of modification sites incorporated into the modifiedamplification product may also vary, although one site is usuallysufficient to significantly reduce contamination. In most cases it is,in fact, preferred to incorporate only one modification site due to theexpected reduction in efficiency of the amplification procedure from,e.g., steric hindrance (caused by the introduction of bulky moieties),interference with hybridization (caused by disruption of hydrogen bodingbetween complementary nucleotides), or lack of complete complementarityof the amplification probes or primers to the target (caused byincorporation of restriction enzyme recognition sites). In this lastcase, however, it will be appreciated that the loss of cyclingefficiency will primarily take place in the first cycle ofamplification, where only amplification sequence having substantialcomplementarity with the modified probe(s) or primer(s) serves astemplate. Thereafter, the expected efficiency loss will diminishproportionately as the relative amount of amplification product actingas template (and having complete complementarity with the probes orprimers) increases.

Where amplification product is not denatured following the final cycleof amplification, it is only necessary to modify one amplification probeor primer. In this case, the resulting modified amplification productwill be double-stranded, enabling both strands to be removed ordestroyed by way of a modification which is incorporated into only onestrand. If, however, the test sample reaction mixture is subjected to adenaturing step following the final amplification cycle, bothamplification primers and at least two opposite strand amplificationprobes (i.e., representing both the "upper" and "lower" strands of theprobe pairs) may have to be modified in order to ensure thatsubstantially all contaminant amplification product is renderednonviable for subsequent hybridization.

Where a ligand has been introduced into the amplification product, theresulting ligand modified amplification product may be selectivelyeliminated by bringing the test sample containing the modifiedamplification product into contact with immobilized specific bindingpartner for the ligand. The immobilizing support is then removed alongwith bound ligand modified amplification product. Where the selectiveelimination is performed on a new test sample (i.e., prior to theinitiation of an amplification procedure) the immobilized specificbinding partner must be brought into contact with the new test samplebefore amplification probe or amplification primer reagents are added tothe new test sample. If the new test sample reaction mixture iscontacted after addition of these reagents, the modified probes orprimers in the reagents will be pulled from solution along with thecontaminant amplification product.

The ligand which is incorporated as the modification will preferably bethe smaller member of a specific binding pair, as this will minimize thereduction in amplification efficiency which is expected because ofsteric hindrance caused by the presence of the ligand on theamplification probes or primers. An example of a preferred ligand isbiotin. Another preferred ligand is fluorescein. The ligand isintroduced into at least one amplification primer, in the case of PCR,or into at least one amplification probe, in the case of LCR. FIG. 1demonstrates a ligand modification of amplification product resultingfrom a ligase chain reaction type of amplification procedure(LCR-derived amplification product) and subsequent removal of themodified amplification product with immobilized specific binding partnerfor the ligand. FIG. 2 shows a similar scheme with respect toamplification product derived from a polymerase chain reaction(PCR-derived amplification product). The resulting biotin- orfluorescein-modified amplification product can be removed fromsubsequent test samples by contacting these samples with immobilizedavidin or anti-fluorescein antibody, respectively.

In the ligand modification embodiment of the invention, it is preferredto modify the amplification probe(s) or primer(s) at a location on theprobe or primer which will not interfere with the action of any enzymeor other reagent used in the amplification procedure; e.g., polymerasein the case of a PCR type of amplification procedure and ligase in thecase of an LCR type of amplification procedure. In situations wherepolymerase can "read through" through the modification site in a PCRtype of amplification procedure, the amplification primer can bemodified at or about any location other than the extending end of theprimer. Where, however, the polymerase cannot read through through themodification site, the PCR primer should be modified at its 5'-end.

Because LCR type amplification procedures do not employ a polymerase,enzyme read through limitations are not a concern in the location ofligand modification sites on the amplification probes. In the case ofLCR, it is generally preferred to modify the amplification probe at anylocation other than the contiguous ends. For amplification probes whichform an end segment of the amplification product, it will also bepossible to modify these probes at their non-ligating ends. Otherwise,all amplification probes will preferably be modified somewhere near thecenter region of the probe.

The amplification probes or primers can be modified with the preferredfluorescein or biotin ligands using methods known to those skilled inthe art. For example, the oligonucleotide probe or primer may bemodified with ligand using a two-step process, wherein an amine group isfirst introduced during synthesis of the oligonucleotide. Followingcoupling, oxidation, deprotection, and removal of the oligo-amine primeror probe from the support used during synthesis, the ligand can beattached.

Another means for controlling carryover contamination from contaminantamplification product involves incorporating a covalently linkedcross-linking agent into at least one of the amplification probes orprimers used in an amplification procedure. The cross-linking agent ofthe resulting modified amplification product may be activated, eitherchemically or photochemically, to covalently cross-link the modifiedamplification product with complementary nucleic acid strands. Thecomplementary nucleic acid strands may be from complementary modified orunmodified amplification product, or they may be in the form of carrierDNA if the modified amplification product has been denatured. In thelatter case, however, both strands of the amplification product willhave had to have been modified for substantially all of the resultingamplification product to be selectively eliminated.

Irreversibly cross-linking modified amplification product will rendermodified amplification product inert to any further amplification bypreventing complete denaturation of the modified complementaryamplification product after this point. In some cases there will be arisk of damaging the target nucleic acid in a new test sample bytreating the sample with the appropriate activator for the cross-linkingagent. This danger can be avoided, however, where the photocross-linking groups have been made to absorb light at a wavelengthwhich is not harmful to nucleic acid targets. Treatment of the new testsamples must, however, proceed in the absence of new amplification probereagents in order to destroy contaminant product without cross-linkingthe new reagents.

The cross-linking agent is incorporated into the amplification probe(s)or primer(s) using methods known to those skilled in the art. In an LCRtype of amplification procedure, it is preferred to locate thecross-linking agent on a middle amplification probe which will, in turn,incorporate the cross-linking agent nearest to the center of theresulting amplification product as is practically possible. With respectto the amplification probe carrying the cross-linking agent, it isfurther preferred to locate the cross-linking agent in the center regionof the probe so as not to interfere with the joining or ligating of theends of the modified probe. This is demonstrated in FIG. 3, wherein thecross-linking modification is incorporated into amplification productfrom one of the middle pair of probes of a three pair set ofamplification probes.

In the case of a PCR type of amplification procedure, it is preferred toincorporate the cross-linking agent as close as possible to theextending end of an amplification primer without interferingsignificantly with the polymerase. As in the case of a ligandmodification, if the presence of a cross-linking agent will interferewith the read through ability of the polymerase used for primerextension, the modification should preferably be placed at the 5'-end ofthe primer. FIG. 4 shows the incorporation of a cross-linkingmodification into PCR-derived amplification product using apresynthesized primer which has been modified at or about the middle ofthe primer.

It is more preferred, however, to modify the amplification product byincorporating at least one enzyme recognition site into theamplification product as the means for controlling carryovercontamination. The enzyme recognition site may be introduced into theamplification product through the use of modified amplification reagents(e.g., modified amplification probes or primers). In PCR typeamplification, it may also be possible to introduce the enzymerecognition site through the use of modified nucleoside triphosphatesthan can serve as both an enzyme recognition site and as a substrate forpolymerases. The enzyme recognition modification site renders theresulting modified amplification product amenable to subsequentdestruction by an enzyme which will selectively cleave the amplificationproduct, but which will leave target or amplification sequence intact.Examples of enzyme recognition sites include, but are not limited to,restriction enzyme recognition sites and RNAse recognition sites.

As with other types of modifications, it is possible to incorporate aplurality of enzyme recognition modification sites into theamplification product. It is, of course, preferred to incorporate theenzyme recognition modification site(s) into the amplification productat a location, or locations, which, when cleaved, will result in themost complete destruction of the amplification product. The preferredplacement for the enzyme recognition site will vary somewhat withrespect to the analyte being sought, and will, in the case ofrestriction enzyme recognition sites be dictated to some degree, by thelocation and number of preferred pseudo restriction sites in the targetsequence. Where only one enzyme recognition site is used to modify theamplification product, it will typically be preferred to place theenzyme recognition site as centrally to the completed amplificationproduct as possible to achieve the most effective destruction.

Central placement of a single enzyme recognition site is more easilyachieved with LCR-derived amplification product than with PCR-derivedamplification product. This is because greater control is available inthe construction of amplification product using presynthesized LCRamplification probes than is available with respect to the predominantportions of a PCR-derived amplification product which are necessarilysynthesized in situ during amplification. For example, with PCR-derivedamplification product, it will typically be necessary to place at leasta part of the single enzyme recognition site on the amplification primerportion of the amplification product, rather than having the entirerestriction site located somewhere in the polymerase extended portionwhich represents the vast central region of the completed amplificationproduct. In contrast, a single enzyme recognition site can be readilyintroduced into the middle of amplification products using an LCR typeof amplification procedure.

Additional limitations present themselves with respect to placement of asingle enzyme recognition site on a PCR-derived amplification product.Most importantly, placement of a single enzyme recognition modificationsite most centrally to the completed amplification product will placethe modification at the extending end of the amplification primer.Because this end of the primer is acted upon by the polymerase to formextension product in the PCR amplification procedure, the placement of amodification site at or near this location could potentially interferewith polymerase extension to form amplification product, and cantherefore decrease the efficiency of amplification where the targetsequence is acting as template sequence. Where, however, multiple enzymerecognition sites are desired, it may be possible to incorporate thesemultiple modification sites into the central (polymerase extended)portion of the resulting amplification product by employing one or moremodified nucleoside triphosphates, such as, for example, aribonucleotide triphosphate (recognized by an RNAse) in place of thecorresponding deoxyribonucleoside triphosphate during amplification.

In order to carry out the method of the present invention using anenzyme recognition modification site recognized by RNAse, a selectedportion of an amplification probe or primer is presynthesized using aseries of RNA bases in place of the DNA bases which form the remainderof the amplification product. The series of RNA bases may be as littleas one RNA base in length (in the case of RNAse A), or several bases inlength (in the case of RNAse H), or the entire length of theamplification product. Unlike the incorporation of restriction enzymerecognition sites, RNAse recognition sites do not necessarily result ina loss of efficiency, because complete complementarity is notnecessarily sacrificed in the latter case. Not only can completecomplementarity exist with RNA base substitutions, but the RNA/DNAhybrid formation can be even stronger than for DNA/DNA hybrids.Preferred location(s) for the series of RNA bases will be apparent tothose skilled in the art through minimal experimentation in light of theteachings of the present invention. It is generally preferred that theseries of RNA bases be at least about one to three bases in length,depending upon the particular RNAse selected as the agent for selectiveelimination of the modified amplification product.

Different types of RNAse enzymes can be used to destroy the RNAserecognition modification site which is incorporated into the modifiedamplification product. A preferred RNAse is RNAse H. RNAse H is specificfor RNA/DNA hybrids and will cut only the RNA bases in the duplex,leaving the DNA strand intact. Where RNAse H is used to destroy enzymerecognition site modified amplification product, it is important thatthe RNAse recognition site be incorporated into both strands of themodified amplification product, and further, that the location of theRNAse recognition sites be at different locations on the amplificationproduct. In this way, the RNAse recognition site on each strand belocated opposite a DNA strand of complementary amplification product,enabling the RNAse H to cut both strands of the modified amplificationproduct duplex. Because the RNAse H enzyme will not cut RNA/RNA orDNA/DNA duplexes, contaminant amplification product which is carriedover into a new test sample can be selectively eliminated without fearof inadvertently destroying DNA target sequence which may hybridize tomodified amplification product.

Other types of preferred RNAse enzymes, reported to be specific forsingle-stranded RNA, include, for example, RNAse A, RNAse CL3, RNAse T₂,and RNAse U₂. Where these types of enzymes are used, there should be nodanger of cutting target DNA. RNAse A is particularly preferred, becauseof its specificity for a single ribonucleotide substitution.

It is also possible to incorporate an enzyme recognition modificationsite that is recognized by a restriction enzyme. Each restriction enzymemodification site on the amplification product will be substantiallycomplementary to a pseudo restriction site on the amplificationsequence. It is preferred that the restriction enzyme site be introducedinto amplification product with a minimal number of mismatches withrespect to the target sequence. It is still more preferred that only onebase be altered to introduce the recognition site; i.e., that therestriction enzyme recognition site be located opposite a preferredpseudo restriction site.

Selection of the restriction enzyme(s) and corresponding restrictionenzyme modification site(s) in a particular amplification product willbe influenced by a number of factors including the costs andavailability of various restriction enzymes. It is also important andpreferred to select an enzyme that has a high cutting efficiency withrespect to the synthetic amplification product that is generated in anamplification procedure. Some restriction enzymes, for example, arebelieved to cleave synthetic oligonucleotide sequences much lessefficiently than wild type sequences. The preferred restrictionenzyme(s) for a given analyte and amplification system will be apparentto those skilled in the art based on the teachings of the presentinvention.

Incorporation of a restriction enzyme modification site into anamplification product is preferably carried out by first selecting fromwithin the target sequence an amplification sequence that contains atleast one preferred pseudo restriction site. Where a number of preferredpseudo restriction sites are contained within the designatedamplification sequence, more options will be available with respect tothe location of the restriction site(s) modification on the modifiedamplification product. Before final selection of the restriction enzymemodification site(s) in the amplification product, it is important toscreen the entire amplification sequence to confirm that there are nonaturally occurring restriction sites in the amplification sequencewhich would be subject to the action of the restriction enzyme chosenfor selective cleavage of the modified amplification product. If suchnaturally occurring sites exist, an alternate restriction enzymemodification site must be chosen, otherwise the amplification sequencewill be destroyed along with the modified amplification product.

In general, restriction enzymes will be able to cleave only modifiedamplification product which is double-stranded. Therefore, therestriction site modified amplification product should not be denaturedbefore contact with the appropriate restriction enzyme. It will,however, be necessary in certain detection systems to denature theamplification product. Where a detection system using complementaryprobes, as disclosed in International Publication No. WO 89/12696, isused it will still be possible to cut the denatured product, as long asthe restriction site is incorporated into the detection probes and theresulting amplification probe/detection probe duplex is not denatured.

Modification of an LCR-derived amplification product with a singlerestriction enzyme modification site is shown in FIG. 5. In thisdiagram, three pairs of probes are ligated to form the amplificationproduct, with the middle pair of probes being provided with therestriction enzyme recognition modification site. This results inLCR-derived modified amplification product which is susceptible to theaction of a restriction enzyme which will destroy the modifiedamplification product by cutting it approximately in half. Theamplification sequence of the target is resistant to the action of theenzyme, and remains in its native state. In this embodiment, however,new test sample must be treated to remove contaminant amplificationproduct before the addition of new probe reagents, because pairs ofmodified amplification probes, necessary for the next amplificationprocedure, will also be destroyed.

FIG. 6 shows the same single site modification incorporated into anamplification primer used in a PCR type of amplification procedure. Theresulting PCR-derived modified amplification product is susceptible todestruction with the same restriction enzyme, but in the case of theresulting PCR-derived amplification product, the cleavage takes placetoward one end of the amplification product. The potential drawback tothis uneven cutting of the PCR-derived product is shown in FIG. 7, whichdemonstrates partial priming of the cleaved PCR-derived amplificationproduct in a subsequent amplification procedure. Partial priming occursbecause the larger portion of the cleaved amplification product containssome of the complementary bases for the primer. Partial priming ofcleaved PCR-derived contaminant amplification product in subsequentamplification procedures can still result in artificially high testsample results, including false positives.

Partial priming can be avoided in some instances if a restriction enzymemodification site can be incorporated sufficiently close to theextending end of the primer that the resulting recognition site actuallyoccurs in the polymerase extended portion of the amplification product.This may, however, be difficult to achieve without interfering with theability of the polymerase to initiate primer extension. Partial primingcan preferably be eliminated by using remote cutting restriction enzymesand incorporating the appropriate corresponding restriction modificationsites into the modified amplification products of a PCR type ofamplification procedure. The incorporation of a remote cuttingrestriction enzyme recognition site into a PCR-derived amplificationproduct is shown in FIG. 8. In this instance, the remote cuttingrestriction enzyme recognition site is incorporated into thepresynthesized amplification primer, but the actual cleavage by therestriction enzyme takes place in the extended portion of the completedamplification product. As a result, the destroyed PCR-derivedamplification product cannot participate in subsequent amplificationevents, because there is no opportunity for partial priming to occur.

Remote cutters can also be useful, and in some cases preferred, in themodification of LCR-derived amplification product. Where the recognitionsite and corresponding cleavage site can be located on regions ofamplification product represented by different pairs of probes, it willbe possible to contact a new test sample with restriction enzyme evenafter the addition of the amplification probe reagents to samplescontaining target without danger of cleaving the probes at the same timeas the contaminant amplification product is destroyed. FIG. 9 shows theincorporation of a remote cutting restriction enzyme recognition siteinto one of the end pairs of a three pair set of probes such that thecompleted LCR-derived amplification product will be cleaved at aposition approximating the middle of the completed product. In thiscase, the cleavage site corresponds to the portion of the completedamplification product represented by the middle pair of amplificationprobes.

It is still further possible to introduce a chemically cleavable siteinto an amplification product by modifying the nucleic acid backbone ofthe amplification probes or primers that are used in an amplificationprocedure. It is more preferred to use this embodiment in an LCR type ofamplification procedure than in a PCR type of amplification procedure,because the presence of a chemically cleavable site is likely tointerfere with the read through ability of some polymerases. Themodified amplification product containing the chemically cleavablemodification site can be destroyed by treatment with a reagent thatcleaves at the modification site(s). The incorporation of a chemicallycleavable moiety may be able to alleviate the problem of reduced cuttingefficiency observed in some of the restriction enzymes with respect tosynthetic sequences, since chemical cleavage reactions are not based ona biologically active enzyme, and therefore do not distinguish betweensynthetic and wild type nucleic acids. Further, most chemicallycleavable moieties will be cut regardless of whether the modifiedamplification product is double-stranded or single-stranded.

In this embodiment, the cleavable sites can be incorporated intopresynthesized probes or primers using commercially available reagentsas shown in FIG. 10. Partial probe or primer sequences are firstsynthesized to contain amine groups on the 3'- or 5'-ends of the partialsequences. These amine-labeled ends are subsequently joined with ahomobifunctional linking reagent to form the complete, but interrupted,sequence which is used as the amplification probe or primer. Forexample, it is possible to use DSP (dithiobis succinimidyl-propionate!),DST (disuccinimidyl-tartarate), or EGS (ethylene glycolbissuccinimidyl-succinate!) (shown in FIG. 10) to join together the 3'- and5'-labeled ends of the partial probe or primer sequences, thus formingthe complete probe reagents containing the cleavable site. In thisinstance, the modified amplification product generated with these probesor primers can be destroyed by cleavage with a reducing agent such asdithiothreitol (where DSP is used as the homobifunctional linkingagent), an oxidizing agent such as sodium periodate (where DST is used),or hydroxylamine (where EGS is used).

It will be preferred to locate the chemically cleavable modificationsite near the middle of an amplification probe or primer so thatdisruption of hybridization will be minimized. It is possible toconstruct the modified amplification probes or primers so that thesereagents are either completely complementary (the cleavable moiety iscontained in a "loop-out") or substantially complementary (the cleavablemoiety is substituted for one of the nucleotides in the probe sequence)to the amplification sequence. In certain instances, such as where aribonucleotide substitution provides the chemically cleavable moiety,the reagents will be completely complementary with the amplificationsequence without the requirement of a loop-out.

Ribonucleotide substitution simultaneously imparts both an enzymerecognition site and a chemically cleavable site to the resultingmodified amplification product through the incorporation of the samelabile bond. This modified amplification product is labile to either orboth: (1) strong base (chemical cleavage); and, (2) certain RNAses(enzymatic destruction). In either case, the resulting cleavage productscan no longer function as templates for amplification.

These labile bonds are preferably incorporated into the modifiedamplification product using amplification probes or primers containing asingle ribonucleotide substitution on their respective 3'-ends. In thecase of PCR, both of the amplification primers will contain theribonucleotide substitution. In the case of LCR, at least one upperstrand and one lower strand amplification probe will contain theribonucleotide substitution. Because the ribonucleotide substitutionoccurs on the 3'-ends of the primers and probes, the labile bonds of themodified amplification product are actually created in situ duringamplification. As a result, the amplification reagents do not containthe labile bond (i.e., are not base labile), thus enabling carryovercontamination to be destroyed by treatment with a strong base or anRNAse in the presence of these treatment-resistant reagents withoutaffecting their integrity to amplify. Furthermore, the ability of thewild type target to serve as a template for amplification is likewiseunaffected by the base or RNAse treatment, enabling destruction of thelabile carryover contaminant amplification product to take place in thepresence of both the target and the amplification reagents in a new testsample.

There are two important requirements for generating PCR- or LCR-derivedmodified amplification product from modified amplification primers orprobes containing a ribonucleotide substitution. First, the enzymerequired for formation of the amplification product must operateeffectively in the presence of the substitution. In the case of PCR,polymerase must extend off of one of the hydroxyl groups on the 3'-endsof the hybridized primers. In the case of LCR, ligase must catalyze thecovalent joining of a 3'-ribose group of one oligonucleotide probe tothe 5'-phosphate group of another oligonucleotide probe. Second, theresulting modified amplification product (which contains one or moreinternal ribonucleotides) must serve as a viable template for subsequentcycles of amplification. (I.e., in the case of PCR, polymerase must readthrough the ribonucleotide linkage.)

The method of the present invention is not limited to LCR and PCR typesof amplification procedures, but can be employed to controlcontamination problems encountered in other types of amplificationprocedures, such as, for example, transcription types of amplificationprocedures and repair chain reaction amplification.

The following examples are provided to aid in the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth, without departing from the spirit of theinvention.

In order to demonstrate the efficacy of the present invention, severaldifferent synthetic nucleic acid sequences were used to simulate: (1)amplification sequences; (2) amplification products; (3) modifiedamplification products; (4) amplification probes; (5) detection probes;(6) amplification primers; (7) detection primers; and, (8) a polylinkerused to determine cutting efficiencies of restriction enzymes onsynthetic nucleic acid sequences. These synthetic sequences are shown inFIGS. 11, 13, 16, 20, 23, and, 28.

EXAMPLE 1 Preparation of Synthetic Sequences

The synthetic amplification sequences (AS), amplification probes (AP),chemically phosphorylated amplification probes (pAP), detection probes(DP), chemically phosphorylated detection probes (pDP) (shown in FIG.11), and the polysite DNA (shown in FIG. 13) were synthesized using anApplied Biosystems model 380B synthesizer (Applied Biosystems, Inc.,Foster City, Calif.), as disclosed in International Publication No. WO89/12696.

Polymer-bound dimethoxytrityl-protected nucleoside (first nucleic acidin sequence) in support columns was first stripped of its5'-dimethoxytrityl protecting group by passing a solution of 3%trichloroacetic acid in dichloromethane through the column for oneminute. The polymer was then washed with acetonitrile, followed byrinsing with dry acetonitrile. The polymer, containing the deprotectednucleoside, was then placed under argon prior to proceeding to the next(condensation) step.

The condensation step was carried out by first treating the polymer withtetrazole in acetonitrile. The polymer-bound deprotected nucleoside wasthen reacted with a protected cyanoethyl nucleoside phosphoramidite(second nucleic acid in sequence; ABI, Foster City, Calif.) inacetonitrile. The condensation reaction was allowed to proceed for 2.0minutes, with the reactants being subsequently removed by filtration.

Condensation was followed by capping the unreacted 5'-hydroxyl groups ofthe nucleosides by passing a solution prepared by mixing one part of amixture available from ABI (Foster City, Calif.) containing aceticanhydride and 2,6-lutidine in THF (tetrahydrofuran) and one part1-methylimidazole in THF (also available from ABI, Foster City, Calif.)through the column for one minute.

Following removal of the capping solution, the polymer was treated for1.5 minutes with an oxidizing solution (0.1M I₂ in H₂O/2,6-lutidine/THF, 1:10:40). This was followed by an acetonitrilerinse. The cycle began again with a trichloroacetic acid-methylenechloride deprotection and was repeated until the desired oligonucleotidesequence was obtained.

The polymer-bound final oligonucleotide chain was treated with freshconcentrated ammonia at room temperature for 2.0 hours. After decantingthe solution from the polymer, the concentrated ammonia solution washeated at 60° C. for 16 hours in a sealed tube.

Each oligonucleotide solution was extracted with 1-butanol and ethylether. The concentration of each extracted solution was determinedspectrophotometrically by measuring absorption at 260 nm. An aliquot ofeach extracted solution containing 5.0 O.D. units of synthesizedoligonucleotide was concentrated for preparative electrophoresis andloaded into a 15% polyacrylamide 7 molar urea gel. Afterelectrophoresis, the product band was visualized by U.V.electrophoresis, cut from the gel, extracted with elution buffer (300 mMsodium acetate (NaOAc), 2.5 mM EDTA, 100 mM Tris.HCl, pH 8.0), and thendesalted on a G-50 Sephadex® (Pharmacia LKB Biotech, Inc., Piscataway,N.J.) column using TEAB eluant (triethyl ammonium bicarbonate) to yieldthe purified oligonucleotide.

The oligonucleotides were chemically phosphorylated using aphosphorylating reagent available from Glen Research Corporation(Herndon, Va.), Catalog No. 10-1900-90. This reagent was first describedby T. Horn and M. Urdea, Tetrahedron Lett., 27, 4705-4708 (1986), andcan be used with standard phosphoramidite automated synthesis protocols.Automated oligonucleotide synthesis on the Applied Biosystemsinstruments proceeded in the 3'→5' direction with the chemicalphosphorylating agent being conveniently introduced at the last cycle ofsynthesis. Phosphorylation efficiency was quantitated through themeasurement of the amount of dimethoxyltrityl group liberated after thelast cycle. Standard deprotection, cleavage, and purification procedureswere used to isolate the desired 5' chemically phosphorylatedoligonucleotides.

EXAMPLE 2 Single Restriction Site Modified Amplification ProductIntroduction of Restriction Site Modification into Amplification Product

This example demonstrates the introduction of restriction enzymemodification sites into amplification products in an LCR type ofamplification procedure. The introduction of restriction enzymemodification sites in this example was accomplished by usingpresynthesized amplification probes containing restriction enzymemodification sites to amplify an amplification sequence containingcorresponding preferred pseudo restriction sites. The restriction enzymemodification sites were selected so that each of the six amplificationprobes (i.e., three pairs) contained a restriction enzyme modificationsite and also contained a single base mismatch with respect to theamplification sequence (i.e., would hybridize opposite a preferredpseudo restriction site in the amplification sequence).

It was also desired to show the selective elimination of restrictionenzyme modified amplification product by demonstrating that theresulting modified amplification products containing the restrictionenzyme modification sites amplify very inefficiently (i.e., aresubstantially destroyed) following treatment with the appropriaterestriction enzyme, while the amplification sequences containing thecorresponding pseudo recognition sites are unaffected followingtreatment with the same restriction enzyme.

Although the modified amplification product (AMP₁) was constructed tocontain one Hae III and two Bbv I restriction endonuclease sites, onlythe Hae III site was used as the selected modification to demonstratecleavage of the amplification product. The amplification sequence (AS)differs from the amplification product with respect to only three basepairs which are introduced into the amplification product (AMP₁) throughthe three pairs of amplification probes (AP₁ /pAP_(1'), pAP₂ /pAP_(2'),and pAP₃ /AP_(3')), each containing one of the three base pairdifferences. The amplification sequence contains the correspondingpseudo restriction sites for the restriction endonuclease sitesincorporated into the amplification product.

A. Restriction Endonuclease Digest of Amplification Sequence, ModifiedAmplification Product, and Carrier DNA

In order to determine the effectiveness of restriction enzyme cleavageof amplification product to destroy its ability to serve as a templatein subsequent amplification procedures, the following restrictionendonuclease reactions were set up in a final volume of 100 μl of buffercontaining: 0.05 mg/ml BSA (bovine serum albumin), 50 mM Tris.HCl, 6.6mM MgCl₂, 6.6 mM DTT (dithiothreitol):

Reaction 1: 2 femtomoles AMP₁ +20 units active Hae III restrictionenzyme

Reaction 2: 2 femtomoles AMP₁ +20 units heat inactivated Hae IIIrestriction enzyme

Reaction 3: 2 femtomoles AS+20 units active Hae III restriction enzyme

Reaction 4: 2 femtomoles AS+20 units heat inactivated Hae IIIrestriction enzyme

Reaction 5: 1 μg human placental DNA+20 units active Hae III restrictionenzyme

Reaction 6: 1 μg human placental DNA+20 units heat inactivated Hae IIIrestriction enzyme

All six reactions were incubated at 37° C. for 16 hours, followed by 90°C. for 10 minutes to destroy any remaining Hae III activity.

B. Amplification of Synthetic Amplification Sequence and RestrictionSite Modified Amplification Product Following Treatment with Active andInactive Rae III Restriction Enzyme

Amplification sequence (AS), restriction enzyme recognition sitemodified amplification product (AMP₁), and human placental DNA (HP-DNA)from the above six reactions were amplified in duplicate in a 15 cycleLCR type amplification procedure using 3 pairs of amplification probes(AP₁ /pAP_(1'), pAP₂ /pAP_(2'), and pAP₃ /AP_(3')), as shown in FIG. 5.It should be noted that these probes will have three base pairmismatches with the amplification sequence, but will be completelycomplementary with AMP₁.

Many different types of ligases can be used to effect ligation of thecontiguously hybridized probes in an LCR type of amplificationprocedure. For example International Publication No. WO 89/12696discloses the use of E. coli ligase (available, e.g., from New EnglandBiolabs, Inc., Beverly, Mass.) to ligate the amplification probes. It isgenerally preferred, however, to use a thermal stable ligase (TSL) inorder to avoid the requirement for continual addition of fresh ligasereagent with each LCR amplification cycle. In this example, thermalstable HB8 DNA ligase isolated from thermus thermophilus (AACC #27634),obtained as a gift from Miho Takahashi (Mitsubishi-Kasei Institute ofLife Sciences, Protein Chemistry Laboratory, 11 Minamiooya, Machida-shi,Tokyo 194, Japan) was used to ligate the amplification probes throughoutthe amplification cycling.

Thermal stable DNA ligase buffer (TSLB) was prepared at 10×concentration to contain 500 mM Tris.HCl (pH 7.6), 66 mM MgCl₂, 10 mMEDTA (ethylenediaminetetraacetic acid), 66 mM DTT, and 500 μg/ml BSA.

Loading Buffer was prepared to contain 11.8 mM EDTA, 6.3M urea, 0.02%bromophenol blue, and 0.02% xylene cyanole.

One hundred attomoles of DNA from each of the reactions 1 through 4 and5 ng of HP-DNA from each of the reactions 5 and 6 were amplified induplicate using a Perkin-Elmer/Cetus Thermocycler (Perkin-ElmerCorporation, Norwalk, Conn.). Each amplification reaction was started ina volume of 50 μl of 1×TSLB containing 2 picomoles of each amplificationprobe (AP₁ /pAP_(1'), pAP₂ /pAP2', and pAP₃ /AP_(3')), 0.03 units ofTSL, and 66 μM NAD in 0.5 ml Eppendorf® tubes. Two drops of mineral oilwere added to each reaction tube to prevent evaporation duringthermocycling. The amplification reactions were cycled 15 times byheating to 90° C. for 2 minutes and 50° C. for 5 minutes for each cycle.

C. Detection of Restriction Site Modified Amplification Product

The amplification products from Example 1B were detected using a twoprobe detection system as described in International Patent ApplicationNo. 89/02649, using the previously described HB8 DNA ligase and TSLBbuffer in place of the E. coli ligase and E. coli ligase buffers ofInternational Patent Application No. 89/02649. The detection probes DP₁and pDP₂, shown in FIG. 11, were used to detect the amplificationproduct.

EDTA/dye reagent was prepared to contain 20 mM EDTA, 6.1M urea, 0.02%bromophenol blue, and 0.02% xylene cyanole.

Capture buffer, 1× in SSPE and 0.01% in ATC (alkaline-treated casein;Livesay, J. H. and R. A. Donald, Clin. Chim. Acta, 123, 193 (1982)), wasprepared by dissolving 8.7 g NaCl, 1.38 g NaH₂ PO₄.H₂ O monobasic, 370mg EDTA, and 100 mg ATC in 800 ml H₂ O. The solution was adjusted to pH6.8 with 5N NaOH, after which the volume was brought up to 1 L.

In order to provide a means for subsequent visualization of the modifiedamplification products by autoradiography, detection probe DP₁ wasphosphorylated with γ³² P-ATP and polynucleotide kinase (BoehringerMannheim Biochemicals, Indianapolis, Ind.) using radioactive phosphorusat a specific activity of approximately 7000 Ci/mmole. Excess γ³² P-ATPwas separated from the phosphorylated oligonucleotide by gel filtrationthrough G50/50 Sephadex® (Pharmacia, Uppsala, Sweden).

The detection reactions were prepared in a final volume of 16 μl of1×TSLB and contained one-tenth of the amplification reaction mixtures,200 femtomoles of each detection probe (γ³² P-DP₁ and pDP₂), 0.03 unitsof TSL, and 66 82 M NAD. The detection reactions were completed byheating to 90° C. for five minutes and then to 50° C. for ten minutes.The reactions were then stopped by addition of an equal volume ofLoading Buffer, followed by heating at 90° C. for an additional threeminutes. The products were separated by electrophoresis of the reactionmixtures on denaturing 15% polyacrylamide gel and visualized byautoradiography. (See FIG. 12.) Relative amplification efficiencies wereestimated based on laser densitometer traces on an LKB UltroScan™ XL(Pharmacia LKB Biotech, Inc., Piscataway, N.J.).

Cleaved amplification product (Reaction 1, Lanes 1 and 2) amplified only7% as efficiently as uncleaved amplification product (Reaction 2, lanes3 and 4). This corresponds to an effective reduction in contaminantamplification product by 93%.

Cleaved (Reaction 3) and uncleaved (Reaction 4) amplification sequence(lanes 5 and 6, and 7 and 8, respectively) amplified with nearlyidentical degrees of efficiency. This demonstrates that amplificationsequence containing pseudo restriction enzyme recognition sites is notaffected by treatment with the same restriction enzyme that destroys thecorresponding modified amplification product.

Cleaved (Reaction 5) and uncleaved (Reaction 6) carrier HP-DNA (lanes 9through 12) showed no signal, indicating that the signals in lanes 1through 8 are not due to any non-specific interactions with DNA or othercomponents in the reaction mixtures.

It should be noted that uncleaved amplification sequence (AS, Reaction4, lanes 7 and 8) amplifies only 18% as efficiently as uncutamplification product (AMP₁, Reaction 2, lanes 3 and 4). This is mostlikely due to the fact that the amplification probes contain threemismatches with respect to the amplification sequence and are completelycomplimentary only to the amplification product.

EXAMPLE 3 Evaluation of Remote Cutting Enzymes

It will be appreciated that the modified amplification product fromExample 2 could also have been cleaved with Bbv I remote cuttingrestriction enzyme. In this case, amplification probes AP₁ /pAP_(1'),pAP₂ /pAP_(2'), and pAP₃ /AP_(3') from Example 2 would not be cleaved bythe Bbv I enzyme. Other remote cutting restriction enzymes can also beused to cleave correspondingly modified amplification product fromeither PCR or LCR types of amplification procedures.

In order to identify remote cutting enzymes with relatively high degreesof cutting efficiencies, eight different enzymes, purchased from NewEngland Biolabs, Inc. (Beverly, Mass.) were selected for evaluation forpotential use as remote cutters in the restriction enzyme recognitionmodification site of the present invention. A 42 base double-strandedsequence of DNA was designed to contain restriction enzyme recognitionsites for all eight of the remote cutting restriction enzymes, as shownin FIG. 13. The upper and lower strands for this polysite DNA sequencewere synthesized using an Applied Biosystems model 380B synthesizer(Applied Biosystems, Inc., Foster City, Calif.), as described inExample 1. These complementary sequences were allowed to hybridize toform the double-stranded sequence for remote cutter evaluation.Equimolar amounts of the upper and lower strands of the polysite DNAwere added together and briefly heated to 90° C. and then allowed tocool to room temperature to form double-stranded DNA suitable forrestriction endonuclease cleavage.

Twenty picomoles of the double-stranded polysite DNA was labeled withγ-³² P at both 5'-ends, as described for γ-³² P-DP₁ in Example 2.

The polysite DNA was then subjected to cleavage by the selected eightrestriction enzymes using eight individual reactions containing 200femtomoles of the ³² P-labeled polysite DNA, 1× buffer, and one of thefollowing enzymes in a total reaction volume of 50 μl:

    ______________________________________             Restriction                      Amount of Restriction    Reaction Enzyme   Enzyme (Units)  Buffer    ______________________________________    1        ALW I    4               1x TSLB    2        Bbv I    2               1x TSLB    3        BSpM I   4               1X TSLB*    4        Fok I    8               1x TSLB    5        Ple I    1               1X TSLB    6        Hga I    8               1x TSLB    7        Hph I    0.8             1x TSLB    8        SfaN I   3               1x TSLB    ______________________________________     *+100 mM NaCl

All eight of the reactions were incubated at 37° C. for 4 hours.Following incubation, one-half of each of the reactions was mixed withan equal volume of EDTA/dye reagent, heated at 90° C. for three minutesto quench the reaction, and then run on a 10% denaturing polyacrylamidegel using standard techniques known in the art and visualized byautoradiography. As shown in FIG. 14, while all of the remote cutterscleaved the modified polysite DNA, Fok I demonstrated the highestcutting efficiency in this system.

EXAMPLE 4 Double Remote Restriction Site Modification of PCR-derivedAmplification Products

Based on the results of the remote cutting enzyme evaluation fromExample 3, a further example was designed to demonstrate the use of FokI as a remote cutting restriction enzyme modification in a PCR type ofamplification procedure. Two Fok I remote cutting restriction enzymemodification sites were introduced into PCR-derived amplificationproduct as illustrated in FIG. 15. These modification sites wereincorporated into the amplification product by using two appropriatelymodified PCR primers, each of which contained a single base mismatchwith respect to a preferred pseudo Fok I restriction site located withinthe amplification sequence. Unlike a non-remote cutter site,incorporation of the remote cutting restriction enzyme recognition siteinto the modified amplification primer results in modified amplificationproduct which contains the Fok I cleavage sites in the extended portionof the amplification product, even though the corresponding recognitionsites lie within the primer-derived portion. Consequently, treatment ofthis modified amplification product with Fok I restriction enzymeresults in cleaved products (as shown in FIG. 15) which are are notsusceptible to the partial priming phenomenon described earlier.

A 147 base pair sequence contained within pUC 9 was selected as theamplification sequence for this example. The selected 147 base pairregion is shown in FIG. 16, along with the sequences of nativeamplification primers AP₆ and AP_(7') (containing no mismatches withrespect to the target) single mismatched amplification primers AP₈ andAP_(9') (to incorporate Fok I restriction enzyme recognition sites intoamplification product) and PAD₂₂, a detection primer.

A. Preparation of pUC 9 Target for Amplification

The 2707 base pair pUC 9 plasmid was used as a source of target DNA forthis example. The pUC 9 target DNA was prepared for amplification bycutting the circular plasmid with Fok I restriction endonuclease toachieve a linear sequence suitable for amplification. It was convenientto use the Fok I restriction enzyme, because it is the same enzyme whichhad already been selected for destruction of the appropriately modifiedamplification product. It is possible, however, to use other restrictionenzymes to linearize or fragment the plasmid, provided an appropriaterestriction site is located within the plasmid. The pUC 9 plasmidcontains five Fok I recognition sites, with amplification sequence beingcontained within the largpat (1340 base pair) Fok I fragment from thelinearization treatment.

It should be noted, that where amplification cycle temperatures remainbelow about 95° C., it will ordinarily be necessary to fragment orlinearize the plasmid, chromosome, or other target in a test sample tofacilitate complete denaturation during the first cycle ofamplification. Where amplification cycle temperatures exceed about 95°C., linearization and/or fragmentation will necessarily occur duringamplification.

The following reagents were used:

Plasmid DNA, pUC 9, at a concentration of 0.45 μg/μl was obtained fromBethesda Research Laboratories (Gaithersburg, Md.).

Reaction Buffer was obtained by a 1/10 dilution of the 10×! reactionbuffer supplied in the GeneAmp™ DNA Amplification Reagent Kit fromPerkin-Elmer/Cetus (Norwalk, Conn.). The 10×! reaction buffer contains100 mM Tris.HCl, 500 mM KCl, 15 mM MgCl₂ and 0.1% (w/v) gelatin. TheReaction Buffer thus obtained for use in this experiment was a 1×!Reaction Buffer.

Fok I restriction endonuclease at a concentration of 4 units/μl wasobtained from New England Biolabs, Inc. (Beverly, Mass.).

The plasmid DNA was cut in the 1×! Reaction Buffer by adding 2 μl (0.9μg) of pUC 9 DNA and 2 μl (8 units) of Fok I restriction endonuclease to50 μl of the 1×Reaction Buffer. The resulting reaction mixture wasallowed to incubate at 37° C. for 45 minutes, with any remaining enzymeactivity being destroyed by heating at 90° C. for 10 minutes followingthe 45 minute incubation period. The concentration of target sequence inthis reaction mixture was calculated at 9.25 femtomoles/μl.

B. PCR Amplification/Detection Using Native Amplification Primers vs.Modified Amplification Primers

In order to determine if the single base mismatches contained withinmodified PCR primers would perform suitably in a PCR type ofamplification procedure, side-by-side amplifications were performedusing either native amplification primers (AP₆ and AP_(7'), havingcomplete complementarity with the amplification sequence) or modifiedamplification primers (AP₈ and AP_(9'), having substantialcomplementarity with the amplification sequence). The side-by-sideamplification reaction mixtures were cycled 20 times, with the productsbeing subsequently visualized using the PAD₂₂ detection primer.

The following reagents used in this example were obtained as part of aPerkin-Elmer/Cetus GeneAmp™ DNA Amplification Reagent Kit: 10 mM dNTP's(DATP, dCTP, dGTP, dTTP), 10×! Reaction Buffer (described in Example4.A., above), and AmpliTaq™ DNA Polymerase (5 units/μl).

Loading Buffer was the same as described in Example 2.B.

Detection primer PAD₂₂ was labeled on its 5'-end with ³² P to a specificactivity of approximately 7000 Ci/mmole, as described in Example 2.C.

Target pUC 9 DNA was prepared by dilution of the Fok I-cut plasmid (fromExample 4.A.) into TE (10 mM Tris, pH 8.0, 0.1 mM EDTA) until thedesired concentration was achieved.

All amplification reactions were run in 0.5 μl Eppendorf® tubes in atotal volume of 50 μl of the 1×Reaction Buffer, which also contained thedNTP's at a concentration of 200 μM and 2.5 units of AmpliTaq™ DNApolymerase. In addition to the above, the reactions also contained:

    ______________________________________             Amplification Primers                             pUC 9 Target    ______________________________________    Reaction 1:               25 pmole each AP.sub.8 and AP.sub.9 '                                 100    amole    Reaction 2:               25 pmole each AP.sub.8 and AP.sub.9 '                                 0.0    amole    Reaction 3:               25 pmole each AP.sub.6 and AP.sub.7 '                                 100    amole    Reaction 4:               25 pmole each AP.sub.6 and AP.sub.7 '                                 0.0    amole    ______________________________________

Two drops of mineral oil were added to each tube to prevent evaporationduring amplification. The reactions were cycled 20 times in aPerkin-Elmer/Cetus Thermocycler by heating to 90° C. for 2 minutes,followed by 5 minutes at 50° C. for each cycle.

The amplification products were selectively visualized using thefollowing polymerase-associated detection system (PAD). In this process,a labeled PAD primer is allowed to hybridize to the resultingamplification product in the presence of an excess of DNPs and apolymerase to generate a labeled extension (detection) product. Thislabeled detection product is then separated from excess labeled PADprimer on PAGE, and subsequently visualized by autoradiography. Becausethe PAD primer must compete with excess amplification primer forhybridization to amplification product, the PAD primer is designed to belonger (i.e., have a higher T_(m)) than the amplification primer. Thus,the reaction temperature can be adjusted to favor hybridization of thePAD primer.

To one tenth (5 μl) of each reaction mixture was added 1 μl of asolution containing 200 femtomoles of ³² P-labeled detection primer(PAD₂₂) and 0.25 units of AmpliTaq™ polymerase. The PAD primer wasannealed and extended by heating the reactions to 90° C. for 5 minutesfollowed by 65° C. for 10 minutes. After cooling to room temperature,the reactions were quenched by the addition of 15 μl of loading bufferfollowed by heating to 90° C. for 5 minutes. The reactions were runalong with a Hpa II-cut pBR 322 ³² P-labeled marker on a 10% denaturingpolyacrylamide gel using standard techniques, and visualized byautoradiography.

The relative intensities of the 147 base pair PAD products for each ofthe reaction mixtures are shown FIG. 17. The modified amplificationprimers, containing the single base mismatch at the pseudo restrictionsite of the amplification sequence, amplified with nearly the sameefficiency as the native primers. (Compare the results from the Reaction1 lane with the Reaction 3 lane.) The results from the zero controls(Reaction 2 and Reaction 4) demonstrate that no perceptible accumulationof product occurs from cycling in the absence of target.

C. Destruction of Modified PCR-derived Amplification Product

The same four reaction mixtures from Example 4.B. were treated witheither active Fok I restriction endonuclease or heat inactivated Fok I(control), and then subjected to PAD detection to evaluate relativecutting efficiencies.

Fok I restriction endonuclease was used to destroy modified PCR-derivedamplification product. All other reagents used in this example wereobtained as part of the Perkin-Elmer/Cetus GeneAmp™ DNA AmplificationReagent Kit as described in Example 4.B.

Heat inactivated Fok I restriction endonuclease (ΔFok I) was prepared byheating the concentrated stock of enzyme to 90° C for 10 minutes. Theresulting ΔFok I was confirmed to be inactive by its observed inabilityto cut plasmid pUC 9 DNA.

A 25 μl aliquot of the 1×Reaction Buffer was added to one-tenth (5 μl)of each Amplification Reaction (1, 2, 3, and 4) from Example 4.B, alongwith either 2 μl (8 units) of active Fok I (Reactions 1A, 2A, 3A, and4A, respectively) or 2 μl of ΔFok I (Reactions 1Δ, 2Δ, 3Δ, and 4Δ,respectively). These reaction mixtures were allowed to incubate for 16hours at 37° C. followed by 90° C. for 5 minutes.

The cutting efficiency of the Fok I and ΔFok I enzymes on thePCR-derived amplification product was evaluated using PAD detection.Following restriction enzyme treatment, a 15 μl aliquot was removed fromeach of the reaction mixtures and then added to 10.5 μl of a solutionwhich was adjusted to be 0.95× in Reaction Buffer and contained 200femtomoles of ³² P-PAD₂₂ (specific activity of 7000 Ci/mmole), 2.5 unitsof AmpliTaq™ DNA polymerase, and was 952 μM in each dNTP (finalconcentration of 392 μM). These reaction mixtures were then heated to90° C. for 5 minutes followed by 65° C. for 10 minutes, in order tocomplete the extension reaction. The reactions were quenched by adding25 μl of loading buffer, followed by heating to 90° C. for 5 minutes.Samples from the quenched reaction mixtures were run on 10%polyacrylamide gel electrophoresis (PAGE), and the products werevisualized by autoradiography. The film was over-exposed during theautoradiography step in order to reveal the presence of cleavageproducts in even trace amounts.

As shown in FIG. 18, the modified amplification product resulting fromthe use of the single base mismatched modified amplification primers(containing Fok I sites) was completely destroyed by treatment withactive Fok I enzyme (Reaction 1A). In contrast, amplification productformed with native primers was unaffected following exposure to the sameactive Fok I enzyme (Reaction 3A), as indicated by the strong PAD signalwhich appears at 147 base pairs on the autoradiogram. As expected,neither the modified nor the unmodified amplification product wasaffected by treatment with heat inactivated Fok I enzyme (Reactions 1Δand 3Δ, respectively). The zero target controls (Reactions 2A, 4A, 2Δ,and 4Δ) did not show any products at 147 base pairs on theautoradiogram, confirming that all observed 147 base pair signals areindeed derived from amplification products.

A slight signal was evident in the PAD detection reaction of Reaction 1Aat about 127 base pairs. This errant signal was determined to arise fromthe PAD primer extending on a modified amplification product templatethat had been cut at only one of the two available Fok I sites asillustrated in FIG. 19. As shown in FIG. 19, there are two possibleproducts that can result from single Fok I cutting, of which only onewill respond to the PAD detection step, producing a 127 base pairproduct. It is important to note that neither of these partially cutmodified amplification products is complementary to both of the modifiedamplification primers which is required for participation as a templatein subsequent amplification procedures.

D. PCR Amplification of Fok I-Treated Amplification Products

In order to further examine the degree of cleavage in the reactions fromExample 4.C., the same reaction mixtures were diluted and subjected to asecond round of 20 cycles of PCR amplification with modifiedamplification primers AP₈ and AP_(9'), as previously described inExample 4.B. The resulting amplification products were then subjected toPAD detection, as described in Example 4.C. In the case of these"re-amplified" samples, even trace amounts of uncut amplificationproduct were expected to be amplified to a detectable level, just ascarryover contaminant product would be expected to produce a detectablesignal in a clinical setting. All of the reagents for there-amplification and subsequent detection procedures were as previouslydescribed.

Each cleavage reaction from Example 4.C. was serially diluted in TE by afactor of 1/100,000. Because the cleavage reactions themselveseffectively resulted in a 1/6 dilution of the original amplificationreaction mixtures, the 1/100,000 dilution represents a total dilution of1/600,000 with respect to the original amplification reaction mixtures.Further, since only 5 μl of the diluted samples were re-amplified,compared with the original volume of 50 μl of amplification reactionmixtures, the re-amplification represents only 1/6,000,000th of thetotal number of amplification products (both modified and unmodified)which were synthesized in situ as a consequence of the original PCRamplification of Example 4.B. This 1/6,000,000 figure is representativeof the level of carryover contamination expected in a typical clinicallaboratory setting.

Five microliters of each of the final dilutions (1/600,000 ) of thecleavage reaction mixtures were added to 45 μl of a solution which wasadjusted such that the final solution, containing the 5 μl of dilutedsample, was 1×in Reaction Buffer, 200 μM in each dNTP, and contained 25picomoles of each amplification primer (AP₈ and AP_(9')), and 2.5 unitsof AmpliTaq™ polymerase. The reaction mixtures resulting from Reactions1A, 2A, 3A, 4A, 1Δ, 2Δ, 3Δ, and 4Δ were designated as Reactions 1A+,2A+, 3A+, 4A+, 1Δ+, 2Δ+, 3Δ+, and 4Δ+, respectively (where "+" indicatesthat the reactions were re-amplified with a second 20 cycles of PCRamplification). Two drops of mineral oil were added to each reactionmixture, after which the 0.5 ml Eppendorf® tubes were sealed and cycled20 times in a Perkin-Elmer/Cetus Thermocycler by heating to 90° C. for 2minutes followed by 50° C. for 5 minutes for each cycle.

A 1 μl aliquot of a solution containing 200 femtomoles of ³² P-labeleddetection primer (PAD₂₂) and 0.25 units of AmpliTaq™ polymerase wasadded to one-tenth (5 μl) of each re-amplified reaction mixture. The PADdetection reaction was run by heating the reaction mixtures to 90° C.for 5 minutes, followed by 65° C. for 10 minutes. The reaction productswere quenched with 15 μl of loading buffer and heated to 90° C. for 5minutes, before running on denaturing 10% PAGE, along with a Hpa II-cutpBR 322 ³² P-labeled marker, and visualized by autoradiography. Aphotograph of the results is shown in FIG. 17. (It should be noted thatthis gel and autoradiogram also contain the data from Reactions 1, 2, 3,and 4 from Example 4.B., above.)

Reactions 1Δ+ and 3Δ+ displayed the types of signals that would beexpected to result from PCR amplification of samples which had beencontaminated with 1/6,000,000th of the products from Reactions 1 and 3,respectively. These results illustrate a typical false positiveresulting from this level of contamination in a 20-cycle PCR type ofamplification. In contrast, amplification of sample contaminated withmodified amplification product which had previously been treated withactive Fok I restriction enzyme (Reaction 1A+) yielded no detectable147-mer product, in other words, no false positive. Treatment of theunmodified amplification product with the Fok I restriction enzyme(reaction 3A+), resulted in a signal of equal intensity compared withtreatment of this same unmodified amplification product with heatinactivated Fok I enzyme (Reaction 3A+). This demonstrates selectivedestruction of the modified amplification product will be by treatmentwith active Fok I restriction endonuclease in the presence of nativetarget sequence, remains unaffected by this treatment. The zero targetamplifications (Reactions 2A+, 2Δ+, 4A+, and 4Δ+) showed no detectionproducts, confirming that the 147-mer products were indeed derived fromamplification product templates, and not spuriously formed.

EXAMPLE 5 Double Remote Restriction Site Modification of PCR-derivedAmplification Products: Cutting After Contamination

While Example 4 demonstrates the successful post-amplification treatmentof a previously amplified test sample to remove carryover contamination,it would, as previously noted, be more effective in a laboratoryenvironment to "pre-treat" new test samples with the appropriate cuttingagent immediately prior to amplification of the new test sample. In thisway, any contaminant amplification product would be destroyed in newsamples by a pre-incubation step which would include addition of thecutting agent (e.g., Fok I) in a sealed tube, with all amplificationreagents present, just before amplification is commenced.

A potential concern with regard to the use of pre-treatmentcontamination control is presented by the expected very low initialconcentration of contaminant amplification product in the new testsamples. This concern arises, because of the observed loss of catalyticactivity of certain cutting agents at these types of low substrateconcentrations. Therefore the effectiveness of the cutting agent atdestroying modified amplification product in new test samples couldseverely minimize the effectiveness of pre-treatment contaminationcontrol. (Example 4 shows that the cutting agent is very effective whenthe modified amplification product is treated with cutting agentimmediately after amplification where the modified product is at arelatively high concentration.)

In order to test whether contamination can be controlled in this manner,a 162 base pair portion of the HIV pol gene was amplified using modifiedamplification primers AP₁₆ and AP_(17') to incorporate Fok I restrictionsites into the amplification products. The product was quantitated bycomparison of the PAD detection signal from extension of detectionprimer PAD₄ on the resulting products to that obtained from standards.The oligonucleotide sequences used in this study are shown in FIG. 20.This modified amplification product was then diluted and added tosubsequent amplification reactions containing native target in order tosimulate a controlled contamination experiment. The resultingcontaminated samples were then treated with either active cutting agent(Fok I) or inaction cutting agent (ΔFok I) prior to amplification.Comparison of the resulting signals provided an indication of theefficiency of destruction of the contaminating product by treatment withthe cutting agent.

A. PCR Amplification of HIV with Modified Amplification Primers andQuantitative Detection of the Modified Product

The nucleic acid used for target and standards in the followingexperiment was a 10.0 kb pBR 322 clone containing approximately 6 kb ofHIV (BH10 isolate; Gallo et al, Nature, 313, 277-284 (1985)). The clonewas linearized for amplification by cutting with BamH I. The same clonewas used to generate standards for the quantitation of product in thedetection step by cutting with Pvu II and Hae III to produce a 255 basepair fragment containing the 162 base pair fragment to be amplified. Thedetection primer PAD₄ should extend on this fragment to product a 192base product. The yield of PCR product can then be estimated bycomparing the resulting 162-mer detection product to the 192-mer productresulting from known amounts of the standard.

The following reagents were obtained as part of a Perkin-Elmer/Cetus(Norwalk, Conn.) GeneAmp™ DNA Amplification Reagent Kit: 10 mM dNTPs(DATP, dCTP, dGTP, and dTTP), and 10×! Reaction Buffer, as described inExample 4.A.

AmpliTaq™ DNA polymerase was obtained from Perkin-Elmer/Cetus at aconcentration of 8 units/μl.

Thermophilic DNA Polymerase was obtained as a gift from MolecularBiology Resources, Inc. (Milwaukee, Wis.) at a concentration of 3units/μl.

Restriction Endonucleases BamH I (25 units/μl), Pvu II (50 units/μl),and Hae III (10 units/μl), and their respective 10× cutting buffers wereobtained from New England Biolabs, Inc. (Beverly, Mass.). The 10×! BamHI buffer was 1500 mM NaCl, 60 mM Tris (pH 7.9), 60 mM MgCl₂, 60 mMβ-mercaptoethanol, and contained BSA at a concentration of 100 μg/ml.The 10×! Hae III buffer was 200 mM Tris (pH 7.9), 100 mM Mg Acetate, 500mM K Acetate, and 10 mM DTT. The 10×! Pvu II buffer was 100 mM Tris (pH7.9), 100 mM MgCl₂, 500 mM NaCl, and 10 mM DTT.

Oligonucleotide Sequences AP₁₆, AP_(17'), and PAD₄ were synthesized andpurified as described in Example 1.

Oligonucleotide PAD₄ was labeled on the 5'-end with ³² P to a specificactivity of approximately 7000 Ci/mmole, as described in Example 2.C.

Human Placental DNA (HP-DNA, Sigma Chemical Company, St. Louis, Mo.) wasused as carrier DNA and was treated by heating a solution at aconcentration of 10 mg/ml in 5 mM MgCl₂ for 10 minutes at 90° C.

Target sequence was prepared by linearizing an HIV clone in pBR 322 (HIV11) containing approximately 5700 base pairs of HIV sequence with BamHI. The clone (0.026 μg) was incubated with 25 units of BamH I in 20 μlof 1×BamH I buffer for 4 hours at 37° C. After cutting, the target wasdiluted such that the desired number of target molecules were containedin 5 μl of TE with HP-DNA present at a concentration of 1 μg/μl.

Standard used to quantitate the amplification products was prepared asfollows: Two μg of the same clone used to prepare target was incubatedin 100 μl of 1×Pvu II cutting buffer containing 250 units of Pvu II for1 hour at 37° C. The solution was adjusted to 0.3 μM in NaCl using 5.0MNaCl and 3 volumes of ethanol was added to precipitate the DNA. Afterspinning at 14,000×g for 30 minutes, the DNA pellet was isolated bydecanting the supernatant and drying the DNA under vacuum. The pelletedDNA was then cut with 50 units of Hae III restriction enzyme in 100 μlof 1×Hae III restriction buffer by incubating for 1 hour at 37° C.Residual enzyme activity was destroyed by heating the reaction to 90° C.for 5 minutes. The resulting 255 base pair restriction fragment,containing the 162 base pair amplification region, should hybridize withthe detection primer PAD₄ and extend in the presence of dNTPs andpolymerase to form a 192 base product that can be used to quantitate the162 base amplification extension product.

All amplification reactions were run in 0.5 ml Eppendorf® tubes in atotal volume of 50 μl of 1×Reaction Buffer, which also contained eachdNTP at a concentration of 200 μM, 25 pmoles of each amplificationprimer (AP₁₆ and AP_(17')), 5.0 μg of HP-DNA, and 2.5 units of AmpliTaq™DNA polymerase. In addition to the above, the reactions also contained:

Reaction 1: 25,000 molecules of target

Reaction 2: 1,000 molecules of target

Reaction 3: 0.0 molecules of target

Two drops of mineral oil were added to each tube to prevent evaporationduring amplification. The reactions were cycled 25 times in aPerkin-Elmer/Cetus Thermocycler by heating to 90° C. for 2 minutes,followed by 5 minutes at 50° C. for each cycle. The amplificationproducts along with standards were selectively visualized using the PAD₄detection primer, as described below.

The detection reactions were run in a total volume of 30 μl of1×Reaction Buffer which also contained each dNTP at a concentration of333 μM, 200 femtomoles of PAD₄ (7000 Ci/mmole), and 0.6 units ofThermophilic (polymerase (MBR). In addition to the above, the detectionreactions also contained:

    ______________________________________                Pvu II/Hae III                            Amplification    Reaction    Cut Standard                            Reaction    ______________________________________    1S          50 femtomoles    2S          40 femtomoles    3S          30 femtomoles    4S          20 femtomoles    5S          10 femtomoles    6S          2.0 femtomoles    1A                      5.0 μl of Reaction 1    2A                      5.0 μl of Reaction 2    3A                      5.0 μl of Reaction 3    ______________________________________

The detection reactions were run by heating the tubes to 90° C. for 5minutes followed by incubation at 60° C. for 10 minutes. The reactionswere quenched by adding 30 μl of loading buffer and heating to 90° C.for 3 minutes followed by cooling to room temperature. The products wereanalyzed by running the samples on 15% denaturing PAGE followed byautoradiography. As shown in the photograph of the autoradiogram in FIG.21, the standards (Lanes 1S-6S) produce a detection product of slightlyslower mobility than the detection product from the amplificationreactions (Lanes 1A-3A). This is consistent with the calculated sizes of192 and 162 base pairs, respectively.

The amplification reactions show a linear response to the amount ofstarting target and the zero target reaction (Lane 3A) does not show anysign of product. Reaction 2 (Lane 2A) can be estimated to containapproximately 10 femtomoles/5 μl of reaction by comparison to the signalproduced from the 10 femtomole standard (Reaction 5S). The modifiedreaction product from this amplification was used in the controlledcontamination experiments detailed below.

B. PCR Amplification of HIV in the Presence of Contamination with andwithout Cutting

In this experiment, wild type target (1000 or 0 molecules) wascontaminated with either 200,000, 20,000, or 0 molecules of modifiedamplification product from Reaction 2A. The reactions were then treatedwith either active cutting agent (Fok I) or inactive cutting agent (ΔFokI). After incubation with the cutting agent in closed reaction tubes fora predetermined time, the reactions were cycled to achieveamplification. The first PCR cycle temperature of 90° C. destroys anyFok I activity, such that the newly formed modified amplificationproducts accumulate exponentially from the uncut wild type targetmolecules.

All reagents used in this example were the same as previously describedin Example 5.A., with the exception of the following additionalreagents. Fok I restriction endonuclease at a concentration of 4units/μl was obtained from New England Biolabs, Inc. (Beverly, Mass.).

The inactive cutting agent, ΔFok I, was obtained by heating a portion ofthe Fok I enzyme in a boiling water bath for 10 minutes.

Loading Buffer was the same as described in Example 2.B.

Modified amplification product from Reaction 2A (starting concentrationof 2 femtomoles/μl), used to contaminate the samples, was seriallydiluted into water to obtain the desired number of contaminatingmolecules for addition to the samples.

All of the reactions were run in 0.5 ml Eppendorf® tubes in a totalvolume of 50 μl of 1×Reaction Buffer, which also contained each dNTP ata concentration of 200 mM, 25 picomoles of each amplification primer(AP₁₆ and AP_(17')), 5.0 μl of HP-DNA and 2.5 units of AmpliTaq™ DNApolymerase. In addition to the above, the reactions also contained:

    ______________________________________               Target   Contamination  Cutting    Reaction   Molecules                        Molecules      Agent    ______________________________________    1          1000     20,000         Fok I    2          1000     20,000         Fok I    3          0        20,000         Fok I    4          0        20,000         Fok I    5          1000     20,000         ΔFok I    6          1000     20,000         ΔFok I    7          0        20,000         ΔFok I    8          0        20,000         ΔFok I    9          1000     200,000        Fok I    10         1000     200,000        Fok I    11         0        200,000        Fok I    12         0        200,000        Fok I    13         1000     200,000        ΔFok I    14         1000     200,000        ΔFok I    15         0        200,000        ΔFok I    16         0        200,000        ΔFok I    17         1000     0              None    18         1000     0              None    19         0        0              None    20         0        0              None    ______________________________________

Reactions designated to include Fok I contained 8 units of the activeenzyme. Reactions designated to include ΔFok I contained an equal amountof the heat inactivated form of the enzyme. Two drops of mineral oilwere added to each tube to prevent evaporation during amplification. Thereactions were placed in a Perkin-Elmer/Cetus thermocycler and heated at37° C. for 60 minutes to complete the cutting reactions followed by 25cycles of heating to 90° C. for 2 minutes and 50° C. for 5 minutes tocomplete the amplification reactions. The reaction products weredetected by combining 5 μl of each reaction with 1 μl of a solution of³² P-labeled PAD₄ in TE (200 femtomoles, 7000 Ci/mmole), followed byheating to 90° C. for 5 minutes, then 60° C. for 10 minutes. Aftercooling to room temperature, 10 μl of loading buffer was added, and thereactions were heated to 90° C. for 3 minutes and again cooled to roomtemperature. The reactions were analyzed by running the samples on a 15%denaturing PAGE followed by autoradiography.

A photograph of the autoradiogram (FIG. 22) shows that contaminatedsamples that were treated with inactive cutting agent prior toamplification give strong false positives (162-mer product) in the zerotarget lanes (reactions 7, 8, 15, and 16). This makes it impossible tosee true positives (reactions 5, 6, 13, and 14) due to the largebackground signals. In contrast, the contaminated samples that weretreated with active cutting agent prior to amplification show strong1000 molecule controls (reactions, 1, 2, 9, and 10) relative to theirrespective zero molecule controls (Reactions 3, 4, 11, and 12). It isinteresting to note that the signals resulting from 1000 molecules oftarget where Fok I was used to cut contamination (reactions 1, 2, 9, and10) are much more pronounced than the signals resulting from 1000molecules of target that were never contaminated or treated with Fok I(Reactions 17 and 18). We have no explanation for this phenomena ofenhanced amplification signals in amplification reactions that containFok I, however, it has been observed routinely.

This mode of contamination control is especially attractive, because theamplification reactions never need to be opened after treatment with thecutting agent in order to begin the amplification reactions. Thisensures that no untreated contamination can enter the reaction vesselprior to amplification to produce false positives.

EXAMPLE 6 Ribonucleotide Modification of PCR-derived AmplificationProduct: Chemical vs. Enzymatic Cleavage of Modified AmplificationProduct

In this example, a single ribonucleotide substitution was made in eachamplification primer in order to introduce a labile modification intoeach strand of the resulting PCR-derived amplification product. Thelabile bonds in the resulting amplification product were created bypolymerase extension off of the modified amplification primers whichcontained the single ribonucleotide on their respective 3'-ends. Thisrendered the modified amplification product amenable to both enzymaticdestruction (with RNAse A at lower temperatures) and chemicaldestruction (in the presence of a strong base at elevated temperatures).

A 75 base pair region of HIV was employed as the amplification sequence.This 75-mer target was amplified using two 15-mer amplification primers(AP₁₈ and AP_(21')) and a 26-mer detection primer (PAD₅), as shown inFIG. 23.

A. PCR Amplification of HIV with Modified Amplification Primers andQuantitative Detection of the Resulting Modified Amplification Product

In this example, an HIV clone pBH10 (Hahn, et al, Nature, 312, 166(1984)) was amplified using the modified amplification primers AP₁₈ andAP_(21') containing a single ribonucleotide modification on their3'-ends. The resulting modified amplification products were thenquantitated by serial dilution and re-amplification concurrently withknown amounts of wild type target standards. These quantitated modifiedamplification product samples were reserved for later use in subsequentexperiments.

AmpliTaq™ DNA Polymerase was obtained from Perkin-Elmer/Cetus at aconcentration of 8 units/μl.

Deoxynucleoside-5'-triphosphates (DATP, dCTP, dGTP, and dTTP) wereobtained as part of the Perkin-Elmer/Cetus GeneAmp™ AmplificationReagent Kit at a concentration of 10 mM each.

Reaction Buffer 10×! contained 100 mM Tris (pH 8.3), 500 mM KCl, 30 mMMgCl₂, and gelatine (Difco Laboratories, Detroit, Mich.) at aconcentration of 1 μg/ml.

Oligonucleotide sequences AP₁₈, AP_(21'), and PAD₅ were synthesized andpurified as described in Example 1, with the exception that AP₁₈ wassynthesized from a RNA support beginning with guanosine, and AP_(21')was synthesized from a RNA support beginning with uracil. These RNAsupports are commercially available from Glen Research Corporation(Herndon, Va.).

Detection oligonucleotide PAD₅ was labeled on the 5'-end with ³² p to aspecific activity of approximately 7000 Ci/mmole, as described inExample 2.C.

Human Placental DNA (HP-DNA, Sigma Chemical Company) was used as carrierDNA, and was treated by heating a solution at a concentration of 10mg/ml in 10 mM MgCl₂ for 10 minutes in a boiling water bath.

Target HIV DNA was prepared by diluting an HIV clone pBH10 in TE to thedesired molecule level and adding an equal volume of a solution of theHP-DNA in TE at a concentration of 2 μg/ml. The presence of HP-DNAprevents non-specific binding of low amounts of target DNA to thecontainer.

All amplification reactions were run in 0.5 ml Eppendorf® reaction tubesin a total volume of 100 μl of 1×Reaction Buffer, which also containedeach dNTP at a concentration of 100 μM, 50 picomoles of eachamplification primer (AP₁₈ and AP_(21')), 5.0 μg of HP-DNA, and 3.2units of AmpliTaq™ polymerase. In addition, each of the reaction vesselsalso contained:

Reaction 1-4: 1000 molecules of target

Reaction 5-6: 0 molecules of target

Two drops of mineral oil were added to each tube to prevent evaporationduring amplification. The reactions were cycled 30 times in aPerkin-Elmer/Cetus Thermocycler by heating to 95° C. for 30 seconds,followed by 50° C. for 5 minutes for each cycle.

After cycling was completed, Reactions 1-4 (1000 molecules of target)were combined, as were Reactions 5 and 6 (0 molecules of target) inorder to provide homogeneous working stocks of both the modifiedamplification product and the corresponding zero molecule control. Thecombined Reactions 1-4 are referred to as Reaction 1K, while thecombined controls, Reactions 5 and 6, are referred to as Reaction 0K.

The amplification products were detected by combining 10 μl of eitherReaction 1K or Reaction 0K with 1 μl of ³² P-labeled detection probePADs (7000 Ci/mmole, 100 femtomoles/μl) and heating to 95° C. for 2minutes, followed by 60° C. for 10 minutes. After cooling to roomtemperature, 10 μl of loading buffer was added and the reactions heatedto 90° C. for 3 minutes, followed again by cooling to room temperature.The reaction products were analyzed by running the samples on 10%denaturing PAGE, followed by autoradiography.

As shown in FIG. 24, there is a very strong detection signal (a 79-mermade up of a 75-mer plus an additional 4 adenosine residues on the PADprimer) from the 1000 molecule reactions (Reaction 1K in Lane 1), and nodetectable signal from the 0 molecule reactions (Reaction 0K in Lane 2).This indicates that this type of modified primer is suitable for PCRtype amplification procedures.

The amount of amplification product was estimated by running a PCRamplification on serial dilutions of Reaction 1K along with knownamounts of wild type target.

All amplification reactions were run in 0.5 ml Eppendorf® tubes in atotal volume of 50 μl of 1×Reaction Buffer, which also contained eachdNTP at a concentration of 200 μM, 25 picomoles of each amplificationprimer (AP₁₈ and AP_(21')), 5.0 μg of HP-DNA, and 3.2 units of AmpliTaq™DNA polymerase. In addition, each of the reactions also contained:

Reaction 7: 1000 molecules of target

Reaction 8: 1000 molecules of target

Reaction 9: 200 molecules of target

Reaction 10: 200 molecules of target

Reaction 11: 0 molecules of target

Reaction 12: 0 molecules of target

Reaction 13: 5 μl of a 1/3×10⁵ dilution of R×n 1K

Reaction 14: 5 μl of a 1/3×10⁵ dilution of R×n 1K

Reaction 15: 5 μl of a 1/3×10⁶ dilution of R×n 1K

Reaction 16: 5 μl of a 1/3×10⁶ dilution of R×n 1K

Reaction 17: 5 μl of a 1/3×10⁷ dilution of R×n 1K

Reaction 18: 5 μl of a 1/3×10⁷ dilution of R×n 1K

Two drops of mineral oil were added to each tube to prevent evaporationduring amplification. Each reaction tube was cycled 30 times in aPerkin-Elmer/Cetus Thermal Cycler by heating to 95° C. for 30 secondsfollowed by 50° C. for 5 minutes for each cycle to achieveamplification. The resulting amplification products were detected bycombining 10 μl from each reaction tube with 1 μl of a solution of ³²P-labeled oligonucleotide PAD₅ (7000 Ci/mmole, 100 femtomoles/μl) andheating to 95° C. for 2 minutes followed by 60° C. for 10 minutes. Aftercooling to room temperature, 10 μl of loading buffer was added to eachreaction and the detection products denatured by heating to 90° C. for 3minutes, followed by cooling to room temperature. The detection productswere visualized by running the samples on 10% denaturing PAGE, followedby autoradiography.

A photograph of the autoradiogram shown in FIG. 25 shows that theproduct yield from the 1/3×10⁷ dilution of Reaction 1K (Lanes 17 and 18)gave a signal of equal intensity to the 1000 molecule wild type standard(Lanes 7 and 8). Thus, Reaction 1K contains 3×10¹⁰ molecules of modifiedamplification product per 5 μl of reaction volume. This would correspondto an average cycle efficiency of 92% for the original amplification.

B. Cutting of Modified Amplification Product with RNAse A

In this experiment, the modified amplification product from Example 6.A.was subjected to RNAse A immediately prior to detection usingoligonucleotide PAD₅. If the ribonucleotide is present in theamplification product, then one would expect to see a detection productwhich is 15 bases shorter than the full length product. Because RNAse Ais specific for ribo-pyrimidines, only the lower strand should becleaved in this reaction (i.e., the lower strand of the modifiedamplification product contains a single uracil ribonucleotide linkage,while the only ribonucleotide component of the upper strand isribo-guanosine, a purine). Further, because the labeled detection primerhybridizes and extends off of the lower strand, the degree of cuttingshould be apparent by the presence of a shorter (64-mer) detectionproduct.

RNAse A was purchased from Sigma Chemical Company and dissolved inTE/150 mM NaCl at a concentration of 10 mg/ml. DNAse activity wasdestroyed by heating this solution for 15 minutes in a boiling waterbath. A 1/200 dilution (50 μg/ml) of this stock into 150 mM NaCl washeated in a boiling water bath for an additional 15 minutes and cooledto room temperature for use in the following experiment.

The detection primer PAD₅ was the same as previously described inExample 6.A.

Reaction 1K and Reaction 0K from the previous experiment were used as asource of modified amplification product.

The cutting reactions were set up as follows:

    ______________________________________    Reaction      Product     RNAse    ______________________________________    1             10 μl Reaction 1K                              1 μl    2             10 μl Reaction 1K                              0 μl    3             10 μl Reaction 0K                              1 μl    4             10 μl Reaction 0K                              0 μl    ______________________________________

The cutting reactions were allowed to proceed for 2 hours at roomtemperature, and the resulting products were detected by adding 1 μl of³² P-labeled PAD₅ (7000 Ci/mmole, 100 femtomoles/μl), followed byheating to 95° C. for 2 minutes and then 60° C. for 10 minutes. Aftercooling to room temperature, 10 μl of loading buffer was added, and thesamples were denatured by heating to 90° C. for 3 minutes, and thencooling to room temperature. The reaction products were then analyzed byrunning the samples on denaturing 10% PAGE, followed by autoradiography.

A photograph of the autoradiogram is shown in FIG. 26. A comparison ofthe RNAse-treated and untreated modified amplification products(Reaction 1, Lane 1, and Reaction 2, Lane 2, respectively) confirms thatthe RNAse is cutting at the ribonucleotide site as is evident by thepresence of the shorter detection product in Lane 1. Furthermore, thereaction appears to be quantitative. As expected, no detection signalsare present in either of the 0 molecule controls (Lanes 3 and 4).

C. PCR Amplification of HIV in the Presence of Contamination with andwithout Strona Base Cuttina In this experiment, wild type targetmolecules (plasmid pBH10) were contaminated with modified amplificationproduct from Reaction 1K and then treated with either KOH (cuttingagent) or KCl (control). After cutting to destroy the contamination, thesamples were neutralized and subjected to PCR amplification to confirmthat the contaminating molecules were destroyed.

Contamination was provided from reaction 1K which contained modifiedamplification product at a concentration of 6×10⁹ molecules/μl, asquantitated in Example 6.A. This sample was serially diluted to obtain aworking stock that contained 1000 molecules of modified amplificationproduct per 5 μl. This working stock also contained HP-DNA at aconcentration of 0.25 μg/μl to prevent loss of product molecules throughnon-specific binding.

Target DNA, HP-DNA, Reaction Buffer, dNTPs, amplification primers AP₁₈and AP_(21'), and labeled detection primer PAD₅ were the same aspreviously described in Example 6.A.

AmpliTaq™ DNA polymerase was obtained from Perkin-Elmer/Cetus at aconcentration of 5 units/μl.

Potassium hydroxide (KOH) was dissolved in deionized water to obtain aworking stock at a concentration of 600 mM.

Potassium chloride (KCl) was dissolved in deionized water to obtain aworking stock at a concentration of 600 mM.

Hydrochloric acid (HCl) was diluted in deionized water to obtain aworking stock at a concentration of 600 mM.

Target molecules were contaminated by mixing 5 μl of target (1000 or 0molecules) with 5 μl of Contamination Working Stock (1000 molecules).These samples were then treated with either 5 μl of KOH or KCl workingstocks (600 mM, with a final concentration of potassium of 200 mM) andheated to 94° C. for 60 minutes to complete the cutting reactions. Thesamples were then neutralized by adding either 5 μl of HCl to theKOH-treated samples or 5 μl of H₂ O to the KCl-treated samples. Thesereactions were designated as follows:

    ______________________________________    Reaction KOH      KCl    HCl     H.sub.2 O                                          Target    ______________________________________    1        0 μl  5 μl                             0 μl 5 μl                                          1000    2        0 μl  5 μl                             0 μl 5 μl                                          1000    3        0 μl  5 μl                             0 μl 5 μl                                          0    4        0 μl  5 μl                             0 μl 5 μl                                          0    5        5 μl  0 μl                             5 μl 0 μl                                          1000    6        5 μl  0 μl                             5 μl 0 μl                                          1000    7        5 μl  0 μl                             5 μl 0 μl                                          0    8        5 μl  0 μl                             5 μl 0 μl                                          0    ______________________________________

All of the reactions were then brought up to a final volume of 100 μland were adjusted to be 1×in Reaction Buffer, and to contain each DNTPat a concentration of 100 μM, 50 picomoles of each amplification primer(AP₁₈ and AP_(21')), and 3.2 units of AmpliTaq™ DNA polymerase. Thereactions were then cycled 30 times in a Perkin-Elmer/Cetus ThermalCycler by heating to 95° C. for 2 minutes, followed by 50° C. for 5minutes for each cycle. The resulting amplification products weredetected by combining 10 μl of each reaction with 1 μl of a solution of³² P-labeled oligonucleotide PAD₅ (7000 Ci/mmole, 100 femtomoles/μl) andheating to 95° C. for 2 minutes, followed by 60° C. for 10 minutes.After cooling to room temperature, 10 μl of loading buffer was added toeach reaction, and the products were denatured by heating to 90° C. for3 minutes and then cooled to room temperature. The products wereanalyzed by running samples on 10% denaturing PAGE, followed byautoradiography.

A photograph of the autoradiogram is shown in FIG. 27. As expected, inthe case where the samples were treated with KCl (Reactions 1-4, Lanes1-4, respectively) the 1000 molecule target reactions cannot bedistinguished. from the 0 molecule target controls. This is because ofthe strong false positive signal resulting from the presence of 1000molecules of contamination that remains unaffected by KCl treatment. Incontrast, in the case where the samples were treated with KOH prior toamplification, the 1000 molecule target amplifications (reactions 5 and6, Lanes 5 and 6, respectively) are easily distinguished from thecorresponding 0 molecule controls (Reactions 7 and 8, Lanes 7 and 8,respectively). This is because the interfering signal from the 1000molecules of contamination was effectively removed by the KOH treatment.

EXAMPLE 7 Ribonucleotide Modification of LCR-derived AmplificationProducts: Chemical and Enzymatic Cleavage of Contamination

In this example, several labile modifications were introduced into eachstrand of an amplification product produced by an LCR type ofamplification. The labile bonds in this example were produced throughthe ligation of amplification probes that contained a singleribonucleotide on their 3'-ends. As with the similarly modifiedPCR-derived amplification product in Example 6, the introduction of thistype of labile bond into the modified amplification product rendered itamenable to both enzymatic destruction (with RNAse A at lowertemperatures) and chemical destruction (in the presence of a strong baseat elevated temperatures).

A 45 base pair region of HIV was employed as the amplification sequencein this example. The 45-mer target was amplified using three pairs ofamplification probes (AP₁₈ /pAP_(18'), pAP₁₉ /pAP_(19'), and pAP₂₀/pAP_(20')), as shown in FIG. 28.

A. LCR Amplification of HIV Using Modified Amplification Probes andQuantitative Detection of the Modified Probes

In this experiment, a 45 base pair region of HIV clone, pBH10 , wasamplified using three pairs of amplification probes (AP₁₈ /pAP_(18'),pAP₁₉ /pAP_(19'), and pAP₂₀ pAP_(20')), as shown in FIG. 28.Amplification probes AP₁₈, pAP₁₉, pAP_(19'), pAP₂₀, and pAP_(20')contained single ribonucleotide residues on their 3'-ends, such thatligation would produce modified amplification product containing labileribonucleotide linkages. The resulting modified amplification productswere then quantitated by comparison to standards and by serial dilution,followed by re-amplification using known amounts of wild type HIV targetas standards. This quantitated amplification product was then used inlater experiments as controlled carryover contamination.

Thermal stable ligase (TSL) from thermus thermophilus, HB8 (ATCC No.27634) and thermal stable DNA ligase buffer (TSLB) was were the same asused in Example 2. B.

Oligonucleotide AP₁₈ was the same as in Example 6.A. and contains aribo-guanosine residue on its 3'-end.

Oligonucleotide pAP_(18'), chemically phosphorylated on its 5'-end, wassynthesized and purified as described in Example 1.

Oligonucleotides pAP₁₉, pAP_(19'), pAP₂₀, and pAP_(20') were synthesizedto contain both a phosphate group on their 5'-ends and a singleribonucleotide residue on their 3'-ends. The phosphate group wasintroduced through chemical phosphorylation, as described in example 1.The 3' ribonucleotides were introduced by initiating synthesis from theappropriate RNA supports (i.e., pAP₁₉ was synthesized from a RNA-Gsupport, pAP_(19') was synthesized from a RNA-C support, pAP₂₀ wassynthesized from a RNA-A support, and pAP_(20') was synthesized from aRNA-U support). The RNA supports were purchased from Glen ResearchCorporation (Herndon, Va.). After synthesis, the oligonucleotides weresubjected to standard deprotection and purification protocols, asdescribed in Example 1. It should be noted that for the followingexperiments, it is not necessary that pAP₂₀ contain a ribonucleotide onits 3'-end, nor is it necessary that oligonucleotide pAP_(20') bephosphorylated on its 5'-end. These modification were incorporated inorder to make the system amenable to the use of a greater number ofamplification probes in future experiments.

Amplification probe pair AP₁₈ /pAP_(18') was ³² P-labeled using γ-³²P-deoxyadenosine triphosphate and T4 kinase to a specific activity ofapproximately 7000 Ci/mmole, as described in Example 2.C. It should benoted that only amplification probe AP₁₈ will be labeled, becauseoligonucleotide pAP_(18') is already phosphorylated on its 5'-end.

HP-DNA was the same as described in Example 6.A.

Target DNA (clone pBH10) was diluted to the appropriate concentrationinto a solution of HP-DNA (1 μg/μl), and then heated in a boiling waterbath for 15 minutes to fragment and denature the plasmid DNA.

All amplification reactions were run in 0.5 ml Eppendorf® tubes in atotal volume of 40 μl of 1×TSLB and contained 2 picomoles of eachamplification probe (AP₁₈, pAP_(18'), pAP₁₉, pAP_(19'), pAP₂₀, andpAP_(20')), an additional 100 femtomoles of ³² P-labeled amplificationpair AP₁₈ /pAP_(18'), 0.06 units of TSL, 10 μg of HP-DNA, and were 66 μMin NAD. Each reaction contained a total of 2.10 picomoles ofamplification pair AP₁₈ /pAP_(18') at an average specific activity of333 Ci/mmole. This label provided a means to visualize the resultingamplification products. In addition to the above, the reactions alsocontained:

Reaction 1: 20 attomoles of target

Reaction 2: 20 attomoles of target

reaction 3: 2 attomoles of target

Reaction 4: 2 attomoles of target

Reaction 5: 0.2 attomoles of target

Reaction 6: 0.2 attomoles of target

Reaction 7: 0.0 attomoles of target

Reaction 8: 0.0 attomoles of target

Two drops of mineral oil were added to each tube to prevent evaporationduring amplification. The reactions were cycled 15 times in aPerkin-Elmer/Cetus Thermal Cycler by heating to 90° C. for 2 minutes,followed by 50° C. for 5 minutes for each cycle. One-eighth of eachreaction (5 μl) was removed and added to 10 μl of loading buffer. Thesamples were denatured by heating to 90° C. for 3 minutes, followed bycooling to room temperature. The amplification reactions were analyzedby running the samples on denaturing 15% PAGE, followed byautoradiography. Approximately 5 minutes before the electrophoreticseparation was complete, known amounts of oligonucleotide pair AP₁₈/pAP_(18') (at the same specific activity used in the experiments) wereloaded on the gel for use as standards in estimating the yield ofamplification product. These standards appear in Lane 9 (25 femtomoles),Lane 10 (2.5 femtomoles), and Lane 11 (0.25 femtomole).

A photograph of the autoradiogram (FIG. 29) shows that the expected45-mer amplification product is formed in direct response to the amountof target present at the beginning of the amplification reaction. Thereis not detectable signal in the zero molecule controls (reactions 7 and8, Lane 7 and 8), while the 45 base amplification products from even thelowest target levels (0.2 attomole, Reactions 5 and 6, Lanes 5 and 6,respectively) were visible from the autoradiogram. Based on comparisonof signals from the 45-mer amplification products in Reactions 1-6(Lanes 1-6) to the standards (Lanes 9-11), the reactions are estimatedto have amplified 10,000 fold. This represents an average cycleefficiency of 85%.

In order to provide a confirmation of the quantity of modifiedamplification product, Reaction 1 was serially diluted (based on theestimated yield of modified amplification product) and re-amplifiedalong with wild type target standards.

All amplification reactions were run in 0.5 μl Eppendorf® tubes in atotal volume of 40 μl of 1×TSLB, and contained 2 picomoles of eachamplification probe (AP₁₈, pAP_(18'), pAP₁₉, pAP_(19'), pAP₂₀, andpAP_(20')), an additional 100 femtomoles of ³² P-labeled amplificationprobe pair AP₁₈ /pAP_(18') (final specific activity of 333 Ci/mmole),and 0.06 units of TSL, 5 μg of HP-DNA, and were 66 μM in NAD. Inaddition to the above, the reactions also contained the following:

    ______________________________________                Target    Modified Amplification    Reaction    (attomoles)                          Product (attomoles)    ______________________________________    12          10        0    13          10        0    14          1         0    15          1         0    16          0         0    17          0         0    18          0         100    19          0         100    20          0         10    21          0         10    22          0         1    23          0         1    ______________________________________

Two drops of mineral oil were added to each reaction tube to preventevaporation during amplification. The reactions were cycled 15 times ina Perkin-Elmer/Cetus Thermal cycler by heating to 90° C. for 2 minutes,followed by 50° C. for 5 minutes for each cycle. One-fourth of eachreaction (10 μl) was removed and added to 10 μl of loading buffer. Thesamples were denatured by heating to 90° C. for 3 minutes, followed bycooling to room temperature. The amplification reactions were analyzedby running the samples on denaturing 15% PAGE, followed byautoradiography.

A photograph of the autoradiogram (FIG. 30) shows that the signalsproduced from the amplification of a calculated 10 attomoles of modifiedamplification product (Reactions 20 and 21, Lanes 20 and 21,respectively) and from 10 attomoles of wild type target (Reactions 12and 13, Lanes 12 and 13, respectively) are equivalent. This confirmsthat the estimated yield of product in Reaction 1 was accurate. Modifiedamplification product from Reaction 1 was used in subsequent examples ascontaminant amplification product.

B. LCR Amplification of HIV in the Presence of Contamination with andwithout Strong Base Cutting

In this example, wild type target molecules (plasmid pBH10) werecontaminated with modified amplification product from Reaction 1, andthen treated with either KOH (cutting agent) or KCl (control). Aftercutting to destroy the contamination, the samples were neutralized andsubjected to LCR amplification to confirm that the contaminatingmolecules were destroyed.

Contamination was provided to the reaction mixtures in the form ofmodified amplification product from Reaction 1 (as quantitated inExample 7.A) to be at a level of 5 femtomoles of product per 1 μl ofreaction mixture. This product was diluted and added to the reactionmixtures at the desired concentration.

Potassium Hydroxide (KOH) was dissolved in deionized water to obtain aworking stock at a concentration of 700 mM.

Potassium Chloride (KCl) was dissolved in deionized water to obtain aworking stock at a concentration of 700 mM.

Hydrochloric Acid (HCl) was dissolved in deionized water to obtain aworking stock at a concentration of 700 mM.

All other reagents were the same as in Example 7.A.

Reactions containing target (10 attomoles or 0 attomoles), Contamination(10 attomoles), HP-DNA (5 μg) and amplification probes (2.0 picomoleseach of AP₁₈ /pAP_(18'), pAP₁₉ /pAP_(19'), and pAP₂₀ /pAP_(20'), and0.10 picomole of ³² P-labeled AP₁₈ /pAP_(18')) in a volume of 18 μl werethen treated with either 4 μl KOH or KCl working stocks (700 mM, with afinal reaction concentration of 127 mM) and heated to 90° C. for 30minutes to complete the cutting reaction. The reactions were thenneutralized with 4 μl of HCl or H₂ O, and subjected to LCR amplificationas described below. The reactions were designated as follows:

    ______________________________________                                          Target    Reaction KOH      KCl    HCl     H.sub.2 O                                          (amoles)    ______________________________________    1        4 μl  0 μl                             4 μl 0 μl                                          10    2        4 μl  0 μl                             4 μl 0 μl                                          10    3        4 μl  0 μl                             4 μl 0 μl                                          0    4        4 μl  0 μl                             4 μl 0 μl                                          0    5        0 μl  4 μl                             0 μl 4 μl                                          10    6        0 μl  4 μl                             0 μl 4 μl                                          10    7        0 μl  4 μl                             0 μl 4 μl                                          0    8        0 μl  4 μl                             0 μl 4 μl                                          0    ______________________________________

All of the reactions were then brought up to a final volume of 40 μl, sothat they were 1×in TSLB, 66 μM in NAD, and contained 0.06 units of TSL.Two drops of mineral oil were added to each reaction to preventevaporation using amplification. The reactions were then cycled 15 timesin a Perkin-Elmer/Cetus Thermal Cycler by heating to 90° C. for 2minutes, followed by 50° C. for 5 minutes for each cycle. One-eighth ofthe reactions (5 μl) was removed and added to 10 μl of loading buffer,and denatured by heating to 90° C. for 3 minutes. The resulting productswere analyzed by running the samples on denaturing 15% PAGE, followed byautoradiography.

A photograph of the autoradiogram (FIG. 31) shows that the contaminatedsamples treated with KCl gave 10 attomole target signals (Reactions 5and 6, Lanes 5 and 6, respectively) that are indistinguishable from thezero attomole target signals (Reactions 7 and 8, Lanes 7 and 8,respectively). This is expected, due to the interfering signal resultingfrom the 10 attomoles of contamination. In contrast, the samples thatwere treated with KOH show no signal from the zero attomole targetreactions (Reactions 3 and 4, Lanes 3 and 4, respectively) and the 10attomole target signals (Reactions 1 and 2, Lanes 1 and 2, respectively)are clearly positive above the zeroes. This demonstrates that the KOHcutting agent effectively eliminates any interfering signals due to thepresence of contamination.

C. LCR Amplification of HIV Followed by Cutting with RNAse A

In this example, a portion of HIV was amplified using the same modifiedamplification probes (FIG. 28) used in the previous examples, with theresulting products being subjected to RNAse A as a cutting agent.Because RNAse A is specific for pyrimidine residues, the treatmentshould cut only the lower strand of the amplification product. The lowerstrand of the resulting modified amplification product contains twointernal ribo-pyrimidine linkages, while the upper strand contains twointernal ribo-purine residues. For this reason, amplification probepAP₂₀ ' was labeled with ³² P such that only the lower strand of theresulting amplification product was labeled.

Because pAP20' already contains a 5'-phosphate group, the ³² P-label wasintroduced by an exchange kination procedure by the following procedure.Ten units of T4 polynucleotide kinase (New England Biolabs, Inc.) wasadded to a solution containing 1.0 picomole of oligonucleotide pAP₂₀ ',2.5 nanomoles of adenosine 5'-phosphate (ADP, Sigma Chemical Company),and 100 picomoles of γ-³² P-adenosine 5'-triphosphate (ATP, 7000Ci/mmole) in 10 μl of buffer (40 mM tris, pH 7.6/10 mM MgCl₂ /12.5 mMDTT). The reaction mixture was allowed to incubate at 37° C. for 30minutes, followed by 90° C. for 5 minutes, to stop the exchangereaction. The labeled oligonucleotide was then separated from excesslabel by passing the reaction through a Sephadex® G-50 column (SigmaChemical Company) using 10 mM triethyl ammonium bicarbonate as aneluent. The fractions containing the oligonucleotide were combined andevaporated using a SpeedVac® concentrator (Savant Instruments, Inc.,Farmingdale, N.Y.). The product was re-suspended in 20 μl of TE (50femtomoles/μl) and determined to have a specific activity ofapproximately 3500 Ci/mmole.

Amplification probes (AP₁₈ /pAP_(18'), pAP₁₉ /pAP_(19'), and pAP₂₀/pAP_(20')), thermal stable ligase (TSL), thermal stable DNA ligasebuffer (TSLB), carrier DNA (HP-DNA, and target DNA (plasmid pBH10) werethe same as describe in Example 7.A.

RNAse A (Sigma Chemical Company) was dissolved in TE/150 mM NaCl at aconcentration of 10 mg/ml. Contaminating DNAse activity was destroyed byheating this solution for 15 minutes in a boiling water bath. A 1/20dilution of this stock in TE was used in the following experiment.

All amplification reactions were run in 0.5 ml Eppendorf® tubes in atotal volume of 20 μl of 1× TSLB and contained 1.0 picomoles of eachamplification probe (AP₁₈, pAP_(18'), pAP₁₉, pAP_(19'), pAP₂₀, andpAP_(20')), and an additional 50 femtomoles of ³² P-labeledamplification probe pAP₂₀ ', 0.03 units of TSL, 5 μg of HP-DNA, and were66 mM in NAD. Each reaction thus contained a total of 1.05 picomoles ofpAP_(20') at a final specific activity of approximately 167 Ci/mmole.This label was provided as a means to visualize the lower strand of theresulting amplification products. In addition to the above, thereactions also contained:

Reaction 1: 0.0 attomoles of target

Reaction 2: 0.0 attomoles of target

Reaction 3: 10.0 attomoles of target

Reaction 4: 10.0 attomoles of target

Two drops of mineral oil were added to each tube to prevent evaporationduring amplification. The reactions were cycled 15 times in aPerkin-Elmer/Cetus Thermal Cycler by heating to 90° C. for 2 minutes,followed by 50° C. for 5 minutes for each cycle. One-fourth (5 μl) ofthe reactions were added to 5 μl of loading buffer, and then denaturedby heating to 90° C. for 3 minutes and cooling to room temperature. Anadditional one-fourth (5 μl) of each of the reactions was removed andtreated with 2 μl of RNAse A solution (1.0 μg). Each of the reactionswas incubated at room temperature for 60 minutes and subsequentlyquenched by the addition of 5 μl of loading buffer to each sample,followed by heating to 90° C. for 3 minutes, and then cooling to roomtemperature. Reactions treated with RNAse A were designated Reactions1R, 2R, 3R, and 4R.

The reaction products were analyzed by running the samples on denaturing10% PAGE followed by autoradiography. A photograph of the autoradiogram(FIG. 32) shows the expected 45-mer amplification product in the 10attomole target reactions (Reactions 3 and 4, Lanes 3 and 4,respectively), while the corresponding zero target controls (Reactions 1and 2, Lanes 1 and 2, respectively) show no sign of amplificationproduct. In contrast, none of the samples treated with RNAse A show anysign of 45-mer amplification product (Reactions 1R, 2R, 3R, and 4R,Lanes 5, 6, 7, and 8, respectively). This confirms that the modifiedamplification product is effectively destroyed by the RNAse A cuttingagent.

What is claimed is:
 1. A method for reducing DNA carryover contaminationin a first test sample which may contain amplification product from apreviously amplified DNA target-containing test sample comprising:(a)amplifying said DNA target of said previous test sample in the presenceof at least one amplification probe or primer modified by the presenceof a series of at least about one to three RNA bases to generate amodified amplification product having an enzyme recognition site orchemically cleavable site not present in said target; and (b) contactingsaid first test sample with a means for cleaving said modifiedamplification product to reduce the amount of modified amplificationproduct from said previous test sample present in said first test samplethereby reducing DNA carryover contamination.
 2. A kit for reducing DNAcarryover contamination in a polymerase chain reaction type ofamplification procedure for amplifying a target sequence comprising:(a)at least one amplification primer modified by the presence of a seriesof RNA bases whereby, during PCR amplification, said modifiedamplification primer produces a modified PCR amplification producthaving an enzyme recognition site or chemically cleavable site notpresent in said target sequence; and (b) a means for cleaving saidmodified PCR amplification product, thereby to reduce DNA carryovercontamination.
 3. A kit for reducing DNA carryover contamination in aligase chain reaction type of amplification procedure for amplifying atarget sequence comprising:(a) at least one amplification probe modifiedby the presence of a series of RNA bases whereby, during LCRamplification, said modified amplification probe produces a modified LCRamplification product having an enzyme recognition site or chemicallycleavable site not present in said target sequence; and (b) a means forcleaving said modified LCR amplification product, thereby to reduce DNAcarryover contamination.
 4. The method of claim 1 wherein said series ofRNA bases is on the 3'-end of said modified amplification probe orprimer.
 5. The method of claim 4 wherein said enzyme recognition site isan RNAse recognition site and said means for cleaving said modifiedamplification product is an RNAse.
 6. The method of claim 5 wherein saidRNAse is selected from the group consisting of RNAse H and RNAse A. 7.The method of claim 6 wherein said RNAse is RNAse H.
 8. The method ofclaim 6 wherein said RNAse is RNAse A and said series of RNA bases isone RNA base in length.
 9. The method of claim 1 wherein saidmodification is a chemically cleavable site and said means for cleavingsaid modified amplification product is a strong base.
 10. The method ofclaim 9 wherein said series of RNA bases is on the 3'-end of saidmodified amplification probe or primer.
 11. The method of claim 10wherein said series of RNA bases is one RNA base in length.
 12. Themethod of claim 9 wherein said strong base is sodium hydroxide.
 13. Thekit of claim 2 wherein said series of RNA bases is at least about one tothree RNA bases in length.
 14. The kit of claim 13 wherein said seriesof RNA bases is on the 3' end of said modified amplification primer. 15.The kit of claim 14 wherein said enzyme recognition site is an RNAserecognition site and said means for cleaving said modified amplificationproduct is an RNAse.
 16. The kit of claim 15 wherein said RNAse isselected from the group consisting of RNAse H and RNAse A.
 17. The kitof claim 16 wherein said RNAse is RNAse H.
 18. The kit of claim 16wherein said RNAse is RNAse A and said series of RNA bases is one RNAbase in length.
 19. The kit of claim 2 wherein said modification is achemically cleavable site and said means for cleaving said modifiedamplification product is a strong base.
 20. The kit of claim 19 whereinsaid series of RNA bases is on the 3'-end of said modified amplificationprobe or primer.
 21. The kit of claim 20 wherein said series of RNAbases is one RNA base in length.
 22. The kit of claim 19 wherein saidstrong base is sodium hydroxide.
 23. The kit of claim 3 wherein saidseries of RNA bases is at least about one to three RNA bases in length.24. The kit of claim 23 wherein said series of RNA bases is on the 3'end of said modified amplification probe.
 25. The kit of claim 24wherein said enzyme recognition site is an RNAse recognition site andsaid means for cleaving said modified amplification product is an RNAse.26. The kit of claim 25 wherein said RNAse is selected from the groupconsisting of RNAse H and RNAse A.
 27. The kit of claim 26 wherein saidRNAse is RNAse H.
 28. The kit of claim 26 wherein said RNAse is RNAse Aand said series of RNA bases is one RNA base in length.
 29. The kit ofclaim 3 wherein said modification is a chemically cleavable site andsaid means for cleaving said modified amplification product is a strongbase.
 30. The kit of claim 29 wherein said series of RNA bases is on the3'-end of said modified amplification probe or primer.
 31. The kit ofclaim 30 wherein said series of RNA bases is one RNA base in length. 32.The kit of claim 29 wherein said strong base is sodium hydroxide.